The spectrum of human herpesvirus 6 infection: from roseola infantum to adult disease.

Human herpesvirus 6 is the causative agent of roseola infantum, a generally benign rash illness of infants. Most persons acquire HHV-6 infection by age 2 years, and HHV-6 infection is a common cause of fever and febrile seizures in infants. In adults, primary infection with HHV-6 can produce a mononucleosis-like illness and, more rarely, severe disease, including encephalitis. In addition to primary infections, HHV-6 can cause clinical illness during reactivation, particularly in immunocompromised persons.

Expression of a pilin subunit BfpA of the bundle-forming pilus of enteropathogenic Escherichia coli in an aroA live salmonella vaccine strain.

Enteropathogenic Escherichia coli (EPEC) is a major cause of childhood diarrhea in developing countries and is a leading cause of severe diarrheal illness among Brazilian infants. As one approach to constructing a vaccine candidate against diarrhea caused by EPEC, we evaluated whether the pilin subunit (BfpA) of the bundle-forming pilus (BFP) could be expressed by a live Salmonella vaccine strain. Several copies of the coding region of BfpA (bfpA) were amplified by PCR from a preparation of the EAF plasmid of EPEC strain B171 and cloned into plasmid vectors. An intact copy of bfpA was subcloned into the heat inducible prokaryotic expression vector pCYTEXP1, and the resulting pBfpA was used to transform the aroA S. typhimurium strain SL3261, generating SL3261(pBfpA). The recombinant vaccine strain was able to express, but not to process, rBfpA as evidenced by a prominent 21 kDa protein that crossreacted with anti-BFP antiserum found only in extracts of heat-treated SL3261(pBfpA), but not in strains of untreated SL3261(pBfpA) or SL3261 not carrying the plasmid. Furthermore, rBfpA accumulation was not toxic to the Salmonella host, as evidenced by similar plating efficiencies between induced and uninduced strains of SL3261(pBfpA). Finally, SL3261(pBfpA) orally administered to BALB/c mice was capable of eliciting a sustained and vigorous humoral immune response to BfpA, achievable even with a single oral dose of approximately 10(9) organisms. Therefore, this pilin product may serve as a potential immunogen as part of a live combined vaccine strategy to prevent two of the major public health problems in Brazil--salmonellosis and EPEC childhood diahrrea.

Tuberculosis among foreign-born persons in New York City, 1992-1994: implications for tuberculosis control.

OBJECTIVE: To study the pattern of transmission of tuberculosis (TB) among foreign-born persons living in New York City. DESIGN: A retrospective multicenter study comparing 158 foreign-born patients to 231 US-born patients diagnosed with TB between 1992 and 1994. The patients were stratified according to their Mycobacterium tuberculosis isolate DNA fingerprint patterns. RESULTS: Nineteen (16%) of 122 isolates from foreign-born TB patients and 75 (42%) of 180 isolates from US-born TB patients had DNA fingerprint patterns (cluster patterns) indicative of recent exogenous transmission (P < 0.001). All cluster pattern strains from foreign-born cases were identical to those found among US-born patients. The likelihood of infection with a cluster pattern strain among foreign-born persons increased with duration of residence in the US, and was significantly associated with being homeless (P < 0.05), or having multidrug-resistant TB (P = 0.00072). CONCLUSION: Although most (84%) cases of TB among foreign-born persons in New York City appear to result from reactivation of infections they acquired abroad, the ones who acquire new infections become infected with strains that are already circulating among the US-born TB patients in New York City, and they have risk factors similar to those faced by US-born tuberculosis patients.

Delivery of human interferon-gamma via gene transfer in vitro: prolonged expression and induction of macrophage antimicrobial activity.

Daily parenteral administration of exogenous interferon-gamma (IFN-gamma) induces or accelerates recovery in experimental and human infections. To develop an alternative delivery system, a replication-defective recombinant adenovirus expressing human IFN-gamma was constructed. The complete coding region of IFN-gamma was amplified by RT-PCR and inserted into an adenovirus cloning vector under the control of a human cytomegalovirus promoter. Recombinant adenovirus containing the IFN-gamma minigene (dAv-IFN-gamma) was isolated from 293 cells co-transfected with the linearized plasmid and an E1 region-deleted fragment of adenovirus genome. Following in vitro infection with dAv-IFN-gamma, dose-dependent and time-dependent expression of IFN-gamma, mRNA and production of soluble protein were demonstrated in human diploid fibroblat and HeLa cell cultures by Northern blot and ELISA, respectively. Extracellular protein secretion persisted for > = 4 weeks following initial transfection, and secreted IFN-gamma induced both antiviral activity (8000-25,000 U/ml) and macrophage activation with killing of intracellular Toxoplasma gondii and leishmania donovani. These results establish that dAv-IFN-gamma generates long-term secretion of biologically active IFN-gamma in vitro and suggest that this vector may be a useful delivery system for cytokine therapy.

Induction of macrophage antiprotozoal activity by monocyte chemotactic and activating factor.

To determine if monocyte chemotactic and activating factor (MCAF) induces intracellular antimicrobial activity, human monocyte-derived macrophages were treated with MCAF and challenged with Toxoplasma gondii and Leishmania donovani. Pretreatment with MCAF induced macrophages to inhibit protozoal replication by approximately 50%. These findings suggest a potential host defense role for MCAF in the inflammatory response to intracellular pathogens.

Multiple host defense defects in failure of C57BL/6 ep/ep (pale ear) mice to resolve visceral Leishmania donovani infection.

Euthymic C57BL/L ep/ep (pale ear [PE]) mice halt the visceral replication of intracellular Leishmania donovani but fail to properly resolve infection. A previous study identified an isolated defect in tissue granuloma formation in these mice; CD4+ and CD8+ cell number, gamma interferon (IFN-gamma) production, and macrophage antimicrobial activity in vitro were all intact. New in vivo results reported here suggest a considerably more complex immune defect, with evidence indicating (i) enhanced control over L. donovani after transfer of normal C57BL/6 spleen cells, (ii) a partially suppressive Th2 cell-associated response mediated by interleukin-4 (IL-4) but not reversed by CD4+ cell depletion, (iii) absent responses to endogenous Th1 cell lymphokines (IFN-gamma and IL-2) but preserved responsiveness to endogenous tumor necrosis factor alpha, (iv) absent responses to exogenous treatment with recognized antileishmanial cytokines (IFN-gamma, IL-2, IL-12, and granulocyte-macrophage colony-stimulating factor [GM-CSF]) not corrected by transfer of C57BL/6 spleen cells, and (v) a deficient response to antimony chemotherapy. Defective hepatic granuloma formation was not corrected by transfer of C57BL/6 spleen cells or by anti-IL-4 administration. While treatment with IL-2 and GM-CSF modified the tissue reaction and induced selected effector cells to encase tissue macrophages, no antileishmanial activity resulted. Together, these observations suggest that the failure of PE mice to resolve visceral L. donovani infection likely represents expression of multiple suboptimal immune responses and/or partial defects, probably involving a combination of T-cell dysfunction, a Th2 cell response, and target cell (macrophage) hyporesponsiveness.

Transmission of multidrug-resistant tuberculosis in a large urban setting.

Multidrug resistance has become an increasingly important problem in the control and prevention of tuberculosis in large urban centers. Although several small outbreaks of multidrug-resistant (MDR) tuberculosis in New York City have been reported, the increase in the number of cases is not fully explained by these recognized outbreaks, and the modes of transmission have not been clearly delineated. Transmission patterns of MDR tuberculosis in New York City, therefore, were studied by stratifying Mycobacterium tuberculosis isolates from 167 newly diagnosed tuberculosis patients according to their DNA restriction fragment length polymorphisms (RFLP). Forty-three (34%) of 127 drug-susceptible isolates and 19 (79%) of 24 multidrug-resistant isolates had RFLP patterns representing possible recent exogenous infection (primary tuberculosis). Patients who had such isolates were more likely to be seropositive for human immunodeficiency virus (58%; p < 0.05), non-Hispanic black (56%; p < 0.005), U.S.-born (57%; p < 0.001), and have MDR tuberculosis (79%; p < 0.0005). In a logistic regression model, primary tuberculosis remained significantly associated with MDR tuberculosis and black race. In contrast to previous reports, in New York City recent exogenous transmission accounts for most new cases of multidrug-resistant tuberculosis.

Double-repetitive-element PCR method for subtyping Mycobacterium tuberculosis clinical isolates.

We describe a rapid method for subtyping Mycobacterium tuberculosis based on PCR amplification of segments located between two distinct DNA repetitive elements. This method, double-repetitive-element PCR, classified 46 clinical isolates as having 25 distinct patterns; the conventional restriction fragment length polymorphism analysis classified the same isolates as having 23 distinct patterns. The double-repetitive-element PCR is a rapid subtyping method that has a discriminating power similar to that of the restriction fragment length polymorphism method.

Effect of granulocyte-macrophage colony-stimulating factor in experimental visceral leishmaniasis.

GM-CSF induces three effects potentially beneficial in visceral leishmaniasis: blood monocyte mobilization, macrophage activation, and amelioration of granulocytopenia. To determine the experimental role and effect of GM-CSF in this intracellular infection, livers from Leishmania donovani-infected BALB/c mice were tested for GM-CSF mRNA expression and mice were treated with anti-GM-CSF antiserum or GM-CSF. L. donovani infection upregulated hepatic GM-CSF mRNA expression by 10-fold, and anti-GM-CSF treatment exacerbated visceral infection and tripled liver parasite burdens 4 wk after challenge. In euthymic mice with established infection, treatment with 1-5 micrograms/d murine GM-CSF induced three dose-related effects: peripheral blood leukocytosis, preferential accumulation of myelomonocytic cells at visceral foci of infection, and leishmanicidal activity comparable to that achieved by IFN-gamma. These effects were either largely or entirely T cell dependent. Treatment with human GM-CSF also induced anti-leishmanial activity but with little effect on peripheral leukocyte number or tissue myelomonocytic cell influx; human G-CSF stimulated marked peripheral granulocytosis and neutrophil tissue accumulation but induced little antileishmanial effect. These results identify a role for endogenous GM-CSF in the initial host defense response to L. donovani, reemphasize the influxing monocyte as an effector cell, and indicate that GM-CSF can be used as an antileishmanial treatment.

Infectious diseases.

Unlike the initial H influenzae type b vaccine, which contained polysaccharide alone, the conjugate vaccines effectively induce immunity in infants as young as 2 months. Multidrug-resistant strains of S pneumoniae are emerging as important pathogens in the United States.

Th1 and Th2 cell-associated cytokines in experimental visceral leishmaniasis.

In experimental Leishmania donovani infection in BALB/c mice, initial susceptibility gives way to T-cell-dependent acquired resistance and eventual control over visceral infection. Since various cytokines appear to underlie the host response to Leishmania infection, we examined infected liver tissue for gene expression of cytokines associated with Th1 (gamma interferon [IFN-gamma] and interleukin-2 [IL-2]) and Th2 cells (IL-4 and IL-10). By Northern (RNA) blot analysis, only IFN-gamma mRNA expression was detected in livers of infected euthymic mice. To determine whether activation of Th1 cells develops selectively in this model, qualitative PCR analysis was used. These results indicated that mRNAs for IFN-gamma, IL-2, IL-4, and IL-10 were all induced by L. donovani infection. The potentially negative Th2 cell-associated response did not appear to play a functional role, however, since resistance was acquired, anti-IL-4 monoclonal antibody treatment did not accelerate control over visceral infection, and serum immunoglobulin E levels remained low. As judged by PCR analysis, IL-4 and IL-10 mRNAs were also expressed under three other conditions without apparent effect: in naive euthymic mice treated with IL-2, which induces leishmanicidal activity; in rechallenged immune mice, which resist reinfection; and in nude mice, which fail to control L. donovani. These results suggest that, like other Leishmania species, L. donovani infection may trigger a potentially suppressive Th2 cell-associated cytokine response. However, in T-cell-intact mice able to control L. donovani, this response either is insufficient to influence outcome or more likely is overshadowed by the Th1 cell response.

Circulating Borrelia burgdorferi in patients with acute Lyme disease: results of blood cultures and serum DNA analysis.

To determine the usefulness of blood culture and polymerase chain reaction (PCR) analysis in detecting circulating Borrelia burgdorferi or its DNA, blood and serum from untreated patients with acute Lyme disease were examined. None of the cultures of blood or serum from the 7 patients tested demonstrated spirochetes. Similarly, all patient serum samples, assayed in two laboratories, were negative for B. burgdorferi DNA using PCR amplification. These results suggest that in patients with acute Lyme disease, spirochetes, spirochete DNA, or both circulate early, only intermittently, or at low levels and that neither culture nor PCR testing of blood or serum, as currently done, appears likely to prove generally useful in the diagnosis of Lyme disease.

Catalase-peroxidase gene sequences in isoniazid-sensitive and -resistant strains of Mycobacterium tuberculosis from New York City.

Isoniazid resistance in Mycobacterium tuberculosis is associated with lack of catalase-peroxidase activity. A recent study showed that some isoniazid-resistant M. tuberculosis strains have a complete deletion of the gene (katG) encoding this enzyme. To examine what proportion of clinical isolates of M. tuberculosis have katG deletion, katG sequences in 80 randomly selected isolates from New York City were analyzed. Polymerase chain reaction was used to amplify a 282-bp segment of M. tuberculosis katG and showed that 35 (90%) of 39 isoniazid-sensitive and 31 (76%) of 41 isoniazid-resistant strains contained katG sequences (P > .1). Ten multidrug and high-level isoniazid-resistant strains with identical restriction fragment length polymorphism patterns were also analyzed. All were found to have katG sequences. These findings suggest that mechanisms other than complete deletion of katG are involved in isoniazid resistance among most clinical isolates of M. tuberculosis from New York City.

Role and effect of IL-2 in experimental visceral leishmaniasis.

In experimental visceral leishmaniasis, acquired resistance is T cell-dependent, involves IFN-gamma-activated macrophages, and is expressed in the tissues by granuloma formation. Resistance also correlates with Ag-stimulated IL-2 secretion; therefore, Leishmania donovani-infected BALB/c mice were treated with anti-IL-2 mAb or rIL-2 to determine the host defense effect of IL-2. In control mice, intracellular hepatic infection peaked at 2 wk and then declined coincident with granuloma development. In contrast, liver parasite burdens in anti-IL-2-treated mice continued to increase until after 4 wk, at which time mature granuloma formation was inhibited. Treatment of mice with continuously administered IL-2 reduced liver burdens by > 50% and led to marked accumulation of granuloma mononuclear cells. The IL-2-responsive mechanism was T cell-dependent and required both L3T4+ and Lyt-2+ cells. IL-2 enhanced IFN-gamma mRNA expression in vivo and was required for IFN-gamma secretion in vitro, and anti-IFN-gamma mAb administration abolished the antimicrobial effect of exogenous IL-2. These results: 1) identify the activity of endogenous IL-2 in both antileishmanial resistance and granuloma formation; 2) demonstrate that exogenous IL-2 can enhance the granulomatous tissue reaction; and 3) indicate that IL-2 treatment stimulates intracellular antimicrobial activity largely via the induction of IFN-gamma.

Transformation-associated cytokine 9E3/CEF4 is chemotactic for chicken peripheral blood mononuclear cells.

9E3/CEF4, which is released from transformed chicken embryo fibroblasts (CEF), is a member of the platelet factor 4 family of inflammatory proteins and may be the avian homolog of interleukin-8. Since the function of 9E3/CEF4 is unknown, we examined the effect of the protein on mitogenicity and chemotaxis, as well as its expression, in fibroblasts and peripheral blood cells. 9E3/CEF4 mRNA was expressed in chicken peripheral blood monocytes, and its expression was stimulated by incubation of the monocytes with lipopolysaccharide or phorbol myristic acetate. Boyden double-membrane analysis of chemotaxis showed that 9E3/CEF4 was chemotactic for chicken peripheral blood mononuclear cells, as well as for heterophils. Untransformed CEF and CEF transformed with Rous sarcoma virus also migrated to 9E3/CEF4 protein, as measured by Boyden single-membrane analysis. 9E3/CEF4 was slightly mitogenic for CEF, causing a doubling of [3H]thymidine uptake when added to serum-starved CEF.9E3/CEF4 was found associated not only with the cell and in the culture medium of Rous sarcoma virus-transformed CEF but also with the extracellular matrix. The in vivo role of 9E3/CEF4 may be involved with chemotaxis and metastasis, rather than with direct stimulation of mitogenicity.

High-resolution analysis of gro alpha mRNA poly(A) shortening: regulation by interleukin-1 beta.

We have previously shown that destabilization of gro alpha mRNA is associated with poly(A) shortening. In this study, we used high-resolution Northern blots to determine the rate and extent of gro alpha mRNA poly(A) shortening. gro alpha mRNA was found to undergo complete deadenylation within 2 h following withdrawal of IL-1. However, the process was not uniform: at 1 h following IL-1 withdrawal, gro alpha mRNA poly(A) lengths ranged from 0 to 180 nucleotides. There was an accumulation of deadenylated gro alpha mRNA which suggested that there may be another step before the mRNA is destroyed. Cycloheximide was found to block gro alpha mRNA degradation at the level of poly(A) shortening. Northern blots revealed a previously unrecognized periodic distribution of poly(A) lengths that was consistent with endonucleolytic cleavage between complexes of poly(A)-binding protein. The findings indicate that the degradation pathway of gro alpha mRNA is a slower version of the c-fos mRNA model, with the important additional feature that deadenylation and degradation are subject to physiologic regulation. This study provides a detailed picture of gro alpha mRNA poly(A) shortening and establishes a basis for further investigation of the mechanism by which IL-1 stabilizes specific mRNAs.