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Biomedical subjects

A L Rothman

Publications and source records attributed to A L Rothman.

At least 37 records · Page 2Linked to original sources
PubMed1998

Predominance of HLA-restricted cytotoxic T-lymphocyte responses to serotype-cross-reactive epitopes on nonstructural proteins following natural secondary dengue virus infection.

We examined the memory cytotoxic T-lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue virus infection. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue virus proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for NS1.2a (NS1 and NS2a collectively) and NS3 proteins, respectively. All CTL lines derived from both patients were cross-reactive with other serotypes of dengue virus. The CD8+ NS1.2a-specific lines from patient KPP94-037 were HLA B57 restricted, and the CD8+ NS3-specific lines from patient KPP94-024 were HLA B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA DR7 restricted. A majority of the CD8+ CTLs isolated from patient KPP94-024 were found to recognize amino acids 221 to 232 on NS3. These results demonstrate that in Thai patients after symptomatic secondary natural dengue infections, CTLs are mainly directed against nonstructural proteins and are broadly cross-reactive.

CD4-Positive T-Lymphocytes↗
PubMed1998

Molecular evolution of dengue type 2 virus in Thailand.

Dengue is a mosquito-borne viral infection that in recent years has become a major international public health concern. Dengue hemorrhagic fever (DHF), first recognized in Southeast Asia in the 1950s, is today a leading cause of childhood death in many countries. The pathogenesis of this illness is poorly understood, mainly because there are no laboratory or animal models of disease. We have studied the genetic relationships of dengue viruses of serotype 2, one of four antigenically distinct dengue virus groups, to determine if viruses obtained from cases of less severe dengue fever (DF) have distinct evolutionary origins from those obtained from DHF cases. A very large number (73) of virus samples from patients with DF or DHF in two locations in Thailand (Bangkok and Kamphaeng Phet) were compared by sequence analysis of 240 nucleotides from the envelope/nonstructural protein 1 (E/NS1) gene junction of the viral genome. Phylogenetic trees generated with these data have been shown to reflect long-term evolutionary relationships among strains. The results suggest that 1) many different virus variants may circulate simultaneously in Thailand, thus reflecting the quasispecies nature of these RNA viruses, in spite of population immunity; 2) viruses belonging to two previously distinct genotypic groups have been isolated from both DF and DHF cases, supporting the view that they arose from a common progenitor and share the potential to cause severe disease; and 3) viruses associated with the potential to cause DHF segregate into what is now one, large genotypic group and they have evolved independently in Southeast Asia for some time.

Dengue↗
PubMed1998

Assessment of human CD4+ and CD8+ T lymphocyte responses in experimental viral vaccine studies.

The traditional in vitro correlate of immunological protection is the induction by a vaccine of neutralizing antibodies against the virus. It was formerly assumed that protection induced by a vaccine was solely due to neutralizing antibodies. Neutralizing antibodies are potent in the prevention of certain diseases, but virus-specific CD4+ T helper cells aid in the proliferation of specific antigen-triggered B cells to make antibodies. CD8+ T cells are responsible for eliminating virus-infected cells during viral illness, and may act as a second line of defence by becoming activated and eliminating any cells that become infected despite the presence of neutralizing antibodies, for example because of a large challenge dose or antigenic variation at the antibody combining sites. We will briefly review our approaches for measuring human virus-specific CD4+ and CD8+ T cell responses to experimental vaccines. It is critically important to have sensitive, quality controlled assays, including positive controls. There are many potential variables in human T cell assays and pitfalls, which usually result in negative CTL results. Uninterpretable data are to be expected unless adequate preliminary testing has been done to establish sensitive, specific and controlled human antigen specific CD4+ and CD8+ T cell assays.

B-Lymphocytes↗
PubMed1997

Herpes virus infection of RPE and MDCK cells: polarity of infection.

Our objective was to determine quantitatively whether herpes simplex virus infects preferentially the apical or basolateral surfaces of two well-differentiated cell types, human retinal pigment epithelial cell and Madin-Darby canine kidney epithelial cells. Secondarily, we sought to localise the mannose 6-phosphate/insulin-like growth factor II receptor, a putative receptor for herpes simplex virus, in the membrane domains of the retinal pigment epithelial cells. Although it has been suggested that receptors utilized by the herpesviruses are heterogeneously distributed on epithelial cells, no quantitative evidence of preferential polarized uptake of wild-type herpes simplex virus into an epithelial cell has yet appeared. Moreover, no evidence has appeared of the distribution of mannose-6-phosphate/insulin-like growth factor II receptor in human retinal pigment epithelial cells. We hypothesized that the preferred pole of uptake and infection by HSV would correlate with the distribution of the receptor. Understanding the preferred site of entry in these cells may shed light on the mechanism of pathological infection and spread of this and related viruses, such as cytomegalovirus, in acute retinal necrosis and herpetic encephalitis. The efficiency of viral infection was assayed two ways. First, using permeable filters on which the monolayer of polarized epithelial cells was grown, we compared the number of foci of infected cells that resulted from an apical infection with that resulting from application of virus to the underside of the filter from which the virus could reach the basolateral surface of the cells. Second, we compared the number of infected cell foci that resulted from an apical infection to the number formed following infection at both the apical and basolateral surfaces of the cells. Both surfaces were exposed to virus following disruption of the tight junctions between cells with a Ca2+ chelator. After the efficiency of infection was normalized for relative surface areas, we found that both cell types were equally infectable with the F strain of the virus. However, there was a difference in the degree of polarized uptake of virus by the two cell types. Virus infected the basolateral surface of the retinal cells only about 6.5 times as effectively as it infected the apical surface of those cells, whereas virus infected the basolateral surface of the kidney epithelial cells about 435 times as effectively as it infected the apical surface of the same cells. These data suggest that herpes simplex virus can efficiently enter either the apical or basolateral surface of retinal pigment epithelial cells, unlike its more polarized preference for the basolateral surface of the kidney epithelial cell type. The mannose 6-phosphate/insulin-like growth factor II receptor was present in human retinal pigment epithelial cells, as determined by Western blotting. Surface biotinylation experiments revealed the presence of the receptor in both the apical and basolateral membranes of the retinal epithelial cells. Our evidence is consistent with the hypothesis that the virus may utilize the mannose 6-phosphate/insulin-like growth factor II receptor to facilitate entry.

Animals↗
PubMed1997

Early clinical and laboratory indicators of acute dengue illness.

A prospective observational study was conducted to identify early indicators of acute dengue virus infection. Children with fever for <72 h without obvious cause were studied at hospitals in Bangkok and Kamphaeng Phet, Thailand, until resolution of fever. Of 172 evaluable subjects (91% of enrollees), 60 (35%) had dengue, including 32 with dengue fever (DF) and 28 with dengue hemorrhagic fever (DHF). At enrollment, children with dengue were more likely than children with other febrile illnesses (OFI) to report anorexia, nausea, and vomiting and to have a positive tourniquet test, and they had lower total white blood cell counts, absolute neutrophil and absolute monocyte counts, and higher plasma alanine and aspartate (AST) aminotransferase levels than children with OFI. Plasma AST levels were higher in children who developed DHF than in those with DF. These data identify simple clinical and laboratory parameters that help to identify children with DF or DHF.

Acute Disease↗
PubMed1997

Dengue in the early febrile phase: viremia and antibody responses.

A multicenter effort was begun in 1994 to characterize the pathophysiology of dengue using a study design that minimized patient selection bias by offering enrollment to all children with undifferentiated fever for <72 h. In the first year, 189 children were enrolled (age range, 8 months to 14 years). Thirty-two percent of these children had dengue infections (60 volunteers). The percentage of children with a secondary dengue infection was 93%, with only 4 (7%) having a primary dengue infection. The virus isolation rate from the plasma of children with dengue was 98%. Viremia correlated highly with temperature. All four dengue virus serotypes were isolated at both study sites. This study demonstrates that all four serotypes of dengue virus can cause dengue hemorrhagic fever, that all dengue patients as defined by serology experience viremia during the febrile phase, and that as fever subsides, so does viremia.

Acute Disease↗
PubMed1997

Rapid diagnosis of dengue viremia by reverse transcriptase-polymerase chain reaction using 3'-noncoding region universal primers.

A reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed as a rapid diagnostic test of dengue viremia. To detect dengue viruses in serum or plasma specimens, a pair of universal primers was designed for use in the RT-PCR. Using these primers, the 3'-noncoding region of dengue virus types 1, 2, 3, and 4 could be amplified, but not those of other flaviviruses, such as West Nile virus, Japanese encephalitis virus, and yellow fever virus, or the alphavirus Sindbis virus. The sensitivity of the RT-PCR assay was similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture. Combining a silica method for RNA isolation and RT-PCR dengue virus could be detected in a 6-hr assay. In a preliminary study using this method, we detected dengue virus in 38 of 39 plasma specimens from which dengue virus had been isolated by mosquito inoculation. We then applied this method for detecting dengue viremia to 117 plasma samples from 62 children with acute febrile illnesses in a dengue-endemic area. We detected dengue viremia in 19 of 20 samples obtained on the day of presentation, which had been confirmed as acute dengue infection by mosquito inoculation and antibody responses. The overall sensitivity of this method was 91.4% (32 of 35; 95% confidence interval [CI] = 82.2-100%). The results from testing plasma samples from febrile nondengue patients showed a specificity of 95.4% (42 of 44; 95% CI = 89.3-100%).

Acute Disease↗
PubMed1996

Dominant recognition by human CD8+ cytotoxic T lymphocytes of dengue virus nonstructural proteins NS3 and NS1.2a.

A severe complication of dengue virus infection, dengue hemorrhagic fever (DHF), is hypothesized to be immunologically mediated and virus-specific cytotoxic T lymphocytes (CTLs) may trigger DHF. It is also likely that dengue virus-specific CTLs are important for recovery from dengue virus infections. There is little available information on the human CD8+ T cell responses to dengue viruses. Memory CD8+CTL responses were analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult volunteers who had received monovalent, live-attenuated candidate vaccines of the four dengue serotypes. All the donors had specific T cell proliferation to dengue and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors, and in the seven donors tested, dengue virus-specific CD8+CTL activity was demonstrated. The nonstructural (NS3 and NS1.2a) and envelope (E) proteins were recognized by CD8+CTLs from six, five, and three donors, respectively. All donors recognized either NS3 or NS1.2a. In one donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL responses using the PBMC of two donors were serotype specific, whereas all other donors had serotype-cross-reactive responses. For one donor, CTLs specific for E, NS1.2a, and NS3 proteins were all HLA-B44 restricted. For three other donors tested, the potential restricting alleles for recognition of NS3 were B38, A24, and/or B62 and B35. These results indicate that the CD8+CTL responses of humans after immunization with one serotype of dengue virus are diverse and directed against a variety of proteins. The NS3 and NS1.2a proteins should be considered when designing subunit vaccines for dengue.

Adult↗
PubMed1996

Limiting vancomycin use to combat vancomycin-resistant Enterococcus faecium.

The implementation, monitoring, and impact of a program to restrict vancomycin use are described. A vancomycin restriction program was implemented in February 1995 at an acute care teaching hospital after guidelines for vancomycin use were established by a multidisciplinary group. Pharmacists reviewed each vancomycin order, suggested alternative treatments when vancomycin use did not comply with the guidelines, and set duration limits for all orders. Orders requiring greater clinical experience for review were referred to a pharmacist or a physician in the division of infectious diseases. The program was monitored by an infectious diseases pharmacist. Data collected after the program was established showed that the volume of vancomycin use decreased substantially. Problems in enforcing the restriction program included administration of vancomycin for surgical prophylaxis before the order reached the pharmacy, continuing use of vancomycin for initial empirical treatment of febrile neutropenic and immunocompromised patients, inadequate tracking of the evaluations, and deficiencies in the evaluations related to a need for continuing education of the pharmacists about the program. Use of vancomycin decreased after a pharmacy-enforced restriction program was implemented.

Anti-Bacterial Agents↗
PubMed1996

Human homolog of patched, a candidate gene for the basal cell nevus syndrome.

The basal cell nevus syndrome (BCNS) is characterized by developmental abnormalities and by the postnatal occurrence of cancers, especially basal cell carcinomas (BCCs), the most common human cancer. Heritable mutations in BCNS patients and a somatic mutation in a sporadic BCC were identified in a human homolog of the Drosophila patched (ptc) gene. The ptc gene encodes a transmembrane protein that in Drosophila acts in opposition to the Hedgehog signaling protein, controlling cell fates, patterning, and growth in numerous tissues. The human PTC gene appears to be crucial for proper embryonic development and for tumor suppression.

Adult↗
PubMed1996

Multiple specificities in the murine CD4+ and CD8+ T-cell response to dengue virus.

The target epitopes, serotype specificity, and cytolytic function of dengue virus-specific T cells may influence their theoretical roles in protection against secondary infection as well as the immunopathogenesis of dengue hemorrhagic fever. To study these factors in an experimental system, we isolated dengue virus-specific CD4+ and CD8+ T-cell clones from dengue-2 virus-immunized BALB/c mice. The T-cell response to dengue virus in this mouse strain was heterogeneous; we identified at least five different CD4+ phenotypes and six different CD8+ phenotypes. Individual T-cell clones recognized epitopes on the dengue virus pre-M, E, NSl/NS2A, and NS3 proteins and were restricted by the I-Ad, I-Ed, Ld, and Kd antigens. Both serotype-specific and serotype-cross-reactive clones were isolated in the CD4+ and CD8+ subsets; among CD8+ clones, those that recognized the dengue virus structural proteins were serotype specific whereas those that recognized the nonstructural proteins were serotype cross-reactive. All of the CD8+ and one of five CD4+ clones lysed dengue virus-infected target cells. Using synthetic peptides, we identified an Ld-restricted epitope on the E protein (residues 331 to 339, SPCKIPFEI) and a Kd-restricted epitope on the NS3 protein (residues 296 to 310, ARGYISTRVEM GEAA). These data parallel previous findings of studies using human dengue virus-specific T-cell clones. This experimental mouse system may be useful for studying the role of the virus serotype and HLA haplotype on T-cell responses after primary dengue virus infection.

Animals↗
PubMed1995

The sick building syndrome and mass hysteria.

Significant overlaps of symptoms in SBS and MH exist including central nervous system manifestations, mucous membrane irritation, skin abnormalities, and eye symptomatology. Both occur with greater frequency in women with lower job rank and in patients with psychological and physical stresses. No specific cause has been identified in over 75% of the cases of SBS. Remotely, less than 25% have been alleged to be secondary to an environmental toxin but even removal of the inciting irritant does not improve the symptoms. Not surprisingly, litigation is lurking in the background with the chronic complainer. There has been no evidence of exposure to toxins or VOCs that exceed the NIOSH safety standards in any SBS cases. The importance of a thorough work up is to distinguish those cases that are secondary to bacteria or toxic contaminants and infection where significant morbidity and death have occurred in the building related illness. Although this is a separate entity it indicates that the building environment can be a significant cause of morbidity and mortality. Other variables such as temperature and humidity do influence the frequency of SBS symptomatology. It may be that we are not yet sophisticated enough to find the causes that are present within our current paradigms. Current levels of toxic data testing may not be sensitive enough to realize that they might be a cause of toxicity. It might be that at low levels with multiple toxins and VOCs present, the additive effects may cause toxic symptomatology whereas individually they do not. In summary, SBS is an emerging phenomena within our litigious society.(ABSTRACT TRUNCATED AT 250 WORDS)

Air Pollution, Indoor↗
PubMed1994

Immunopathologic mechanisms of dengue hemorrhagic fever and dengue shock syndrome.

Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas of the world. The immunopathological mechanisms that result in severe complications of dengue virus infection, i.e. dengue hemorrhagic fever (DHF), are important to determine. Primary dengue virus infections induce serotype-specific and serotype-cross-reactive, CD4+ and CD8+ memory cytotoxic T lymphocytes (CTL). In secondary infections with a virus of a different serotype from that which caused primary infections, the presence of cross-reactive non-neutralizing antibodies results in an increased number of infected monocytes by dengue virus--antibody complexes. This in turn results in marked activation of serotype cross-reactive CD4+ and CD8+ memory CTL. We hypothesize that the rapid release of cytokines and chemical mediators caused by T cell activation and by CTL-mediated lysis of dengue virus-infected monocytes triggers the plasma leakage and hemorrhage that occurs in DHF.

Cross Reactions↗
PubMed1993

Dengue virus protein recognition by virus-specific murine CD8+ cytotoxic T lymphocytes.

The identification of the protein targets for dengue virus-specific T lymphocytes may be useful for planning the development of subunit vaccines against dengue. We studied the recognition by murine dengue virus-specific major histocompatibility complex class I-restricted, CD8+ cytotoxic T lymphocytes (CTL) of dengue virus proteins using recombinant vaccinia viruses containing segments of the dengue virus genome. CTL from H-2k mice recognized a single serotype-cross-reactive epitope on the nonstructural (NS) protein NS3. CTL from H-2b mice recognized a serotype-cross-reactive epitope that was localized to NS4a or NS4b. CTL from H-2d mice recognized at least three epitopes: a serotype-specific epitope on one of the structural proteins, a serotype-cross-reactive epitope on NS3, and a serotype-cross-reactive epitope on NS1 or NS2a. Our findings demonstrate the limited recognition of dengue virus proteins by CTL from three inbred mouse strains and the predominance of CTL epitopes on dengue virus nonstructural proteins, particularly NS3. Since human dengue virus-specific CTL show similar patterns of recognition, these findings suggest that nonstructural proteins should be considered in designing vaccines against dengue.

Animals↗
PubMed1991

Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities.

Epidermolysis bullosa simplex (EBS) is characterized by skin blistering due to basal keratinocyte fragility. In one family studied, inheritance of EBS is linked to the gene encoding keratin 14, and a thymine to cytosine mutation in exon 6 of keratin 14 has introduced a proline in the middle of an alpha-helical region. In a second family, inheritance of EBS is linked to loci that map near the keratin 5 gene. These data indicate that abnormalities of either of the components of the keratin intermediate filament heterodipolymer can impair the mechanical stability of these epithelial cells.

Base Sequence↗
PubMed1990

Human immune responses to dengue viruses.

Dengue fever (DF) and dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) are major public health problems in many areas of the world. We are analyzing the human immune responses to dengue viruses, in order to understand the mechanism of recovery from dengue virus infections and the pathogenesis of DHF/DSS. Human natural killer (NK) cells lyse dengue virus-infected cells to a greater degree than uninfected cells. Antibodies to dengue viruses augment the lysis of dengue virus-infected cells by NK cells. Dengue virus-infected monocytes produce high levels of interferon alpha (IFN alpha). DR+ lymphocytes also produce high levels of IFN alpha after contact with dengue virus-infected monocytes. The IFN alpha produced protects uninfected monocytes from dengue virus infection. These results suggest that NK cells and IFN alpha may play an important role in controlling primary dengue virus infection. Dengue virus-specific CD4+CD8(-)T lymphocytes and CD4(-)CD8+T lymphocytes are present in the peripheral blood mononuclear cell population from donors who were infected with dengue virus. Most of CD4+T lymphocytes are dengue serotype-crossreactive. They lyse dengue virus-infected autologous cells in an HLA class II-restricted fashion, and produce interferon gamma (IFN gamma). IFN gamma augments dengue virus infection of monocytic cells in the presence of antidengue virus antibodies by increasing the number of Fc gamma receptors. Dengue virus-specific CD8+T lymphocytes lyse dengue virus-infected autologous cells in an HLA class I-restricted fashion. These CD8+T lymphocytes are also dengue serotype-crossreactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Differentiation, T-Lymphocyte↗