Influence of age, social patterns and nasopharyngeal carriage on antibodies to three conserved pneumococcal surface proteins (PhtD, PcpA and PrtA) in healthy young children.

The acquisition of specific antibodies is paramount to protect children against pneumococcal diseases, and a better understanding of how age, ethnicity and/or Streptococcus pneumoniae (Spn) nasopharyngeal carriage influence the acquisition of antibodies to pneumococcal surface proteins (PSP) is important for the development of novel serodiagnostic and immunisation strategies. IgG antibody titres against three conserved PSP (PhtD, PcpA and PrtA) in the sera of 451 healthy children aged 1 to 24 months from Israel [Jewish (50.1 %) and Bedouin (49.9 %)] were measured by enzyme-linked immunosorbent assay (ELISA), while nasopharyngeal swabs from these children were assessed for the presence of Spn. Globally, anti-PhtD and anti-PrtA geometric mean concentrations (GMC; EU/ml) were high at <2.5 months of age [PhtD: 35.3, 95 % confidence interval (CI) 30.6-40.6; PrtA: 71.2, 95 % CI 60-84.5], was lower at 5-7 months of age (PhtD: 10, 95 % CI 8-12.4; PrtA: 17.9, 95 % CI 14.4-22.1) and only increased after 11 months of age. In contrast, an increase in anti-PcpA was observed at 5-7 months of age. Anti-PcpA and anti-PrtA, but not anti-PhtD, were significantly higher in Bedouin children (PcpA: 361.6 vs. 226.3, p = 0.02; PrtA: 67.2 vs. 29.5, p < 0.001) in whom Spn nasopharyngeal carriage was identified earlier (60 % vs. 38 % of carriers <6 months of age, p = 0.002). Spn carriage was associated with significantly higher anti-PSP concentrations in carriers than in non-carriers (p < 0.001 for each PSP). Thus, age, ethnicity and, essentially, nasopharyngeal carriage exert distinct cumulative influences on infant responses to PSP. These specific characteristics are worthwhile to include in the evaluation of pneumococcal seroresponses and the development of new PSP-based vaccines.

Synthetic collagen fascicles for the regeneration of tendon tissue.

The structure of an ideal scaffold for tendon regeneration must be designed to provide a mechanical, structural and chemotactic microenvironment for native cellular activity to synthesize functional (i.e. load bearing) tissue. Collagen fibre scaffolds for this application have shown some promise to date, although the microstructural control required to mimic the native tendon environment has yet to be achieved allowing for minimal control of critical in vivo properties such as degradation rate and mass transport. In this report we describe the fabrication of a novel multi-fibre collagen fascicle structure, based on type-I collagen with failure stress of 25-49 MPa, approximating the strength and structure of native tendon tissue. We demonstrate a microscopic fabrication process based on the automated assembly of type-I collagen fibres with the ability to produce a controllable fascicle-like, structural motif allowing variable numbers of fibres per fascicle. We have confirmed that the resulting post-fabrication type-I collagen structure retains the essential phase behaviour, alignment and spectral characteristics of aligned native type-I collagen. We have also shown that both ovine tendon fibroblasts and human white blood cells in whole blood readily infiltrate the matrix on a macroscopic scale and that these cells adhere to the fibre surface after seven days in culture. The study has indicated that the synthetic collagen fascicle system may be a suitable biomaterial scaffold to provide a rationally designed implantable matrix material to mediate tendon repair and regeneration.

Failure to elicit seroresponses to pneumococcal surface proteins (pneumococcal histidine triad D, pneumococcal choline-binding protein A, and serine proteinase precursor A) in children with pneumococcal bacteraemia.

Pneumococcal surface proteins (PSPs) elicit antibody responses in infants and young children exposed to Streptococcus pneumoniae. These seroresponses could contribute to the aetiological diagnosis of pneumococcal disease, e.g. during the clinical development of novel PSP-based vaccines. In this study, we assessed the kinetics of antibody responses to three highly conserved and immunogenic PSPs (pneumococcal histidine triad D (PhtD), pneumococcal choline-binding protein A (PcpA), and serine proteinase precursor A (PrtA)) in 106 children (median age, 21.3 months; males, 58.5%) admitted for pneumococcal bacteraemia. Anti-PhtD, anti-PcpA and anti-PrtA antibodies were measured by ELISA, and compared in 61 pairs of acute (≤7 days) and convalescent (>14 days of admission) serum samples. Acute serum titres were similar to those observed in healthy children, and were unaffected by the acid dissociation of circulating immune complexes. Despite proven bacteraemia, seroresponses (≥2-fold increase in anti-PSP antibody concentrations) were only identified in 31 of 61 children (50.8%), directed against PrtA (n = 23, 37.7%), PcpA (n = 19, 31.1%), and PhtD (n = 16, 26.2%), or several PSPs (two PSPs, n = 13, 21.3%; three PSPs, n = 7, 11.5%). Certain seroresponses were very strong (maximal fold-increases: PhtD, 26; PcpA, 72; PrtA, 12). However, anti-PSP antibody concentrations failed to increase in the convalescent sera of 30 of 61 (49.2%) bacteraemic children, and even declined (≥2 fold) in 13 of 61 (21.3%), mostly infants aged <6 months (8/13, 61.5%), possibly through consumption of maternal antibodies. Thus, pneumococcal bacteraemia may fail to elicit antibody responses, and may even have an antibody-depleting effect in infants. This novel observation identifies an important limitation of serology-based studies for the identification of bacteraemic children.

Immunity to pneumococcal surface proteins in children with community-acquired pneumonia: a distinct pattern of responses to pneumococcal choline-binding protein A.

The aetiological diagnosis of community-acquired pneumonia (CAP) is challenging in children, and serological markers would be useful surrogates for epidemiological studies of pneumococcal CAP. We compared the use of anti-pneumolysin (Ply) antibody alone or with four additional pneumococcal surface proteins (PSPs) (pneumococcal histidine triad D (PhtD), pneumococcal histidine triad E (PhtE), LytB, and pneumococcal choline-binding protein A (PcpA)) as serological probes in children hospitalized with CAP. Recent pneumococcal exposure (positive blood culture for Streptococcus pneumoniae, Ply(+) blood PCR finding, and PSP seroresponse) was predefined as supporting the diagnosis of presumed pneumococcal CAP (P-CAP). Twenty-three of 75 (31%) children with CAP (mean age 33.7 months) had a Ply(+) PCR finding and/or a ≥ 2-fold increase of antibodies. Adding seroresponses to four PSPs identified 12 additional patients (35/75, 45%), increasing the sensitivity of the diagnosis of P-CAP from 0.44 (Ply alone) to 0.94. Convalescent anti-Ply and anti-PhtD antibody titres were significantly higher in P-CAP than in non P-CAP patients (446 vs. 169 ELISA Units (EU)/mL, p 0.031, and 189 vs. 66 EU/mL, p 0.044), confirming recent exposure. Acute anti-PcpA titres were three-fold lower (71 vs. 286 EU/mL, p <0.001) in P-CAP children. Regression analyses confirmed a low level of acute PcpA antibodies as the only independent predictor (p 0.002) of P-CAP. Novel PSPs facilitate the demonstration of recent pneumococcal exposure in CAP children. Low anti-PcpA antibody titres at admission distinguished children with P-CAP from those with CAP with a non-pneumococcal origin.

Procedural learning and dyslexia.

Three major 'neural systems', specialized for different types of information processing, are the sensory, declarative, and procedural systems. It has been proposed (Trends Neurosci., 30(4), 135-141) that dyslexia may be attributable to impaired function in the procedural system together with intact declarative function. We provide a brief overview of the increasing evidence relating to the hypothesis, noting that the framework involves two main claims: first that 'neural systems' provides a productive level of description avoiding the underspecificity of cognitive descriptions and the overspecificity of brain structural accounts; and second that a distinctive feature of procedural learning is its extended time course, covering from minutes to months. In this article, we focus on the second claim. Three studies-speeded single word reading, long-term response learning, and overnight skill consolidation-are reviewed which together provide clear evidence of difficulties in procedural learning for individuals with dyslexia, even when the tasks are outside the literacy domain. The educational implications of the results are then discussed, and in particular the potential difficulties that impaired overnight procedural consolidation would entail. It is proposed that response to intervention could be better predicted if diagnostic tests on the different forms of learning were first undertaken.

FOXP3 gene expression in a tuberculosis case contact study.

Regulatory T lymphocytes (T(regs)) that express FOXP3 are involved in the beneficial attenuation of immunopathology, but are also implicated in down-regulation of protective responses to infection. Their role in tuberculosis (TB) is unknown. We classified 1272 healthy TB contacts according to their tuberculin skin test (TST) and interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) results and 128 TB cases, and studied the expression of FOXP3 and interleukin (IL)-10 in blood samples. Compared to the uninfected contact group (TST(-), ELISPOT(-)), we observed higher levels of FOXP3 mRNA in blood from TB patients (< 0.001), but IL-10 expression was slightly lower (P = 0.04). In contrast, FOXP3 expression levels were significantly lower (P = 0.001) in the recently infected contacts (TST(+), ELISPOT(+)) but there was no difference for IL-10 (P = 0.74). We hypothesize that during early/subclinical TB, most of which will become latent, FOXP3(+) T(regs) may be sequestered in the lungs, but when TB becomes progressive, FOXP3 reappears at increased levels in the periphery. While these findings do not reveal the role, beneficial or harmful, of T(regs) in TB, they emphasize the probable importance of these cells.

Rising ELISPOT count prior to the onset of symptoms of full-blown tuberculosis disease.

The relationship between the T-cell response to mycobacterial antigens and the likelihood of progression to disease has not been defined. We report a rapidly rising ELISPOT count in a 55-year-old man with evidence of Mycobacterium tuberculosis infection prior to the onset of symptoms of disease. This case illustrates the possible utility of quantitative changes in the ELISPOT count in predicting progression from M. tuberculosis infection to disease.

Identifying ELISPOT and skin test cut-offs for diagnosis of Mycobacterium tuberculosis infection in The Gambia.

SETTING: An urban area, The Gambia. OBJECTIVE: To identify ELISPOT and PPD skin test cut-offs, targeting sensitivity and specificity equivalence. DESIGN: Tuberculosis cases >5 years of age and their household contacts underwent ELISPOT, HIV and PPD skin tests. Cases and contacts sleeping in a different house were used to estimate sensitivity and specificity, providing two planes for estimating cut-offs. Specificity was adjusted for infection from previous exposure using a multivariate discrimination algorithm. RESULTS: The point on the line of intersection of the planes that maximised sensitivity and specificity equivalence occurred at 4 spots (95% confidence interval [CI] 3.5-5, multiplier=0 ) for CFP-10 and 5.5 spots (4.5-8, multiplier=0 for ESAT-6), yielding a sensitivity and specificity of 76% for both antigens. Combining ESAT-6 and CFP-10 using an 'or' statement yielded a maximum equivalence sensitivity and specificity of 76.5% at 6 spots for ESAT-6 and 11.5 spots for CFP-10. For the PPD skin test sensitivity and specificity, an equivalence of 78% occurred at 11 mm induration (9-13 mm). CONCLUSION: An ELISPOT cut-off for ESAT-6 or CFP-10 could be set at 4-8 spot forming units (20-40 spots per million), with little benefit from combining the results. A cut-off of 9-13 mm for the PPD skin test is reasonable when comparing with the ELISPOT.

Mycobacterium tuberculosis genome-wide screen exposes multiple CD8 T cell epitopes.

Mounting evidence suggests human leucocyte antigen (HLA) class I-restricted CD8(+) T cells play a role in protective immunity against tuberculosis yet relatively few epitopes specific for the causative organism, Mycobacterium tuberculosis, are reported. Here a total genome-wide screen of M. tuberculosis was used to identify putative HLA-B*3501 T cell epitopes. Of 479 predicted epitopes, 13 with the highest score were synthesized and used to restimulate lymphocytes from naturally exposed HLA-B*3501 healthy individuals in cultured and ex vivo enzyme-linked immunospot (ELISPOT) assays for interferon (IFN)-gamma. All 13 peptides elicited a response that varied considerably between individuals. For three peptides CD8(+) T cell lines were expanded and four of the 13 were recognized permissively through the HLA-B7 supertype family. Although further testing is required we show the genome-wide screen to be feasible for the identification of unknown mycobacterial antigens involved in immunity against natural infection. While the mechanisms of protective immunity against M. tuberculosis infection remain unclear, conventional class I-restricted CD8(+) T cell responses appear to be widespread throughout the genome.

Design and implementation of relational databases relevant to the diverse needs of a tuberculosis case contact study in the Gambia.

The data requirements of a large multidisciplinary tuberculosis case contact study are complex. We describe an ACCESS-based relational database system that meets our rigorous requirements for data entry and validation, while being user-friendly, flexible, exportable, and easy to install on a network or stand alone system. This includes the development of a double data entry package for epidemiology and laboratory data, semi-automated entry of ELISPOT data directly from the plate reader, and a suite of new programmes for the manipulation and integration of flow cytometry data. The double entered epidemiology and immunology databases are combined into a separate database, providing a near-real-time analysis of immuno-epidemiological data, allowing important trends to be identified early and major decisions about the study to be made and acted on. This dynamic data management model is portable and can easily be applied to other studies.

Performance of a rapid phage-based test, FASTPlaqueTB, to diagnose pulmonary tuberculosis from sputum specimens in South Africa.

SETTING: Twelve primary health care clinics in the South Peninsula Administration, Cape Town, Western Cape Province, South Africa. OBJECTIVE: To evaluate the performance of FAST-PlaqueTB, a new phage-based test, for the rapid diagnosis of TB in individuals with no previous history of TB treatment presenting at primary health care clinics in Cape Town, South Africa. DESIGN: A comparative study of FASTPlaqueTB, auramine smear microscopy and Lowenstein-Jensen culture of 1692 decontaminated sputum specimens from 853 patients suspected of having TB. Resolution of discrepant results was undertaken by review of clinical information, chest X-ray and follow-up of treatment outcomes. RESULTS: FASTPlaqueTB detected TB in 75.2% of culture-confirmed cases and 70.3% of all cases with a clinical diagnosis of TB, with a specificity of 98.7% and 99.0%, respectively. The performance parameters of FASTPlaqueTB were significantly superior to those of concentrated auramine smear microscopy (63.4% and 61.3% sensitivity, and 97.4% and 97.3% specificity in culture-confirmed and all cases, respectively). Of those patients with two negative sputum smears, FAST-PlaqueTB detected TB in 54.1% of TB cases confirmed by culture and 48.8% of all cases with a clinical diagnosis of TB. A combination of smear microscopy and FASTPlaqueTB enabled 81.2% of culture-confirmed cases and 78.4% of total TB cases to be detected within 2 days of presentation. CONCLUSION: FASTPlaqueTBTM is a rapid, manual test for the diagnosis of TB. The test has significantly higher sensitivity overall compared with auramine sputum smear microscopy in individuals with no previous history of TB treatment, although smear microscopy did detect the most infectious of the TB cases. The FAST-PlaqueTB test is easy to perform, requires no dedicated equipment, and results are read by eye within 48 hours. This test can be useful for the diagnosis of TB in developing countries with a high burden of TB where other rapid diagnostic tests may not be appropriate. The test shows promising performance, particularly in the diagnosis of smear-negative disease, and could be used in conjunction with smear microscopy to aid in the diagnosis of additional cases of TB.

Mitochondrial phylogeography and population history of pine martens Martes martes compared with polecats Mustela putorius.

The flora and fauna of Europe are linked by a common biogeographic history, most recently the Pleistocene glaciations that restricted the range of most species to southern refugial populations. Changes in population size and migration, as well as selection, have all left a signature on the genetic differentiation. Thus, three paradigms of postglacial recolonization have been described, inferred from the patterns of DNA differentiation. Yet some species, especially wide-ranging carnivores, exhibit little population structuring between the proposed refugia, although relatively few have been studied due to the difficulty of obtaining samples. Therefore, we investigated mitochondrial variation in pine martens, Martes martes, in order to understand the extent to which they were affected by glacial cycles, and compared the results with an analysis of sequences from polecats, Mustela putorius. A general lack of ancient lineages, and a mismatch distribution that is consistent with an expanding population, is evidence that the present-day M. martes and Mu. putorius in central and northern Europe colonized from a single European refugium following a recent glaciation. There has also been interspecific mitochondrial introgression between M. martes and the sable M. zibellina in Fennoscandia.

Enhanced immunogenicity of CD4(+) t-cell responses and protective efficacy of a DNA-modified vaccinia virus Ankara prime-boost vaccination regimen for murine tuberculosis.

DNA vaccines whose DNA encodes a variety of antigens from Mycobacterium tuberculosis have been evaluated for immunogenicity and protective efficacy. CD8(+) T-cell responses and protection achieved in other infectious disease models have been optimized by using a DNA immunization to prime the immune system and a recombinant virus encoding the same antigen(s) to boost the response. A DNA vaccine (D) and recombinant modified vaccinia virus Ankara (M) in which the DNA encodes early secreted antigenic target 6 and mycobacterial protein tuberculosis 63 synthesized, and each was found to generate specific gamma interferon (IFN-gamma)-secreting CD4(+) T cells. Enhanced CD4(+) IFN-gamma T-cell responses were produced by both D-M and M-D immunization regimens. Significantly higher levels of IFN-gamma were seen with a D-D-D-M immunization regimen. The most immunogenic regimens were assessed in a challenge study and found to produce protection equivalent to that produced by Mycobacterium bovis BCG. Thus, heterologous prime-boost regimens boost CD4(+) as well as CD8(+) T-cell responses, and the use of heterologous constructs encoding the same antigen(s) may improve the immunogenicity and protective efficacy of DNA vaccines against tuberculosis and other diseases.

Human CD8+ CTL specific for the mycobacterial major secreted antigen 85A.

The role of CD8(+) CTL in protection against tuberculosis in human disease is unclear. In this study, we stimulated the peripheral blood mononuclear cells of bacillus Calmette-Guérin (BCG)-vaccinated individuals with live Mycobacterium bovis BCG bacilli to establish short-term cell lines and then purified the CD8(+) T cells. A highly sensitive enzyme-linked immunospot (ELISPOT) assay for single cell IFN-gamma release was used to screen CD8(+) T cells with overlapping peptides spanning the mycobacterial major secreted protein, Ag85A. Three peptides consistently induced a high frequency of IFN-gamma responsive CD8(+) T cells, and two HLA-A*0201 binding motifs, P(48-56) and P(242-250), were revealed within the core sequences. CD8(+) T cells responding to the 9-mer epitopes were visualized within fresh blood by ELISPOT using free peptide or by binding of HLA-A*0201 tetrameric complexes. The class I-restricted CD8(+) T cells were potent CTL effector cells that efficiently lysed an HLA-A2-matched monocyte cell line pulsed with peptide as well as autologous macrophages infected with Mycobacterium tuberculosis or recombinant vaccinia virus expressing the whole Ag85A protein. Tetramer assays revealed a 6-fold higher frequency of peptide-specific T cells than IFN-gamma ELISPOT assays, indicating functional heterogeneity within the CD8(+) T cell population. These results demonstrate a previously unrecognized, MHC class I-restricted, CD8(+) CTL response to a major secreted Ag of mycobacteria and supports the use of Ag85A as a candidate vaccine against tuberculosis.

In vivo administration of O(6)-benzylguanine does not influence apoptosis or mutation frequency following DNA damage in the murine intestine, but does inhibit P450-dependent activation of dacarbazine.

Clinically relevant cancer chemotherapeutic alkylating agents such as temozolomide and dacarbazine induce apoptosis and are mutagenic via the formation of O(6)-alkylguanine adducts in DNA. The DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) functions by dealkylating such adducts and can thus prevent apoptosis and mutagenesis. In attempts to maximize the clinical effectiveness of these alkylating agents, inhibitors of AGT such as O(6)-benzylguanine (BeG) have been developed. We show here that within murine small intestinal crypt cells, BeG administration does not alter the apoptotic response to the direct-acting methylating agents N-methyl-N-nitrosurea (MNU), temozolomide and N-methyl-N'-nitro-N-nitrosoguanidine. Furthermore, we show that BeG pretreatment fails to elevate the mutation frequency at the murine Dlb-1 locus following exposure to MNU. Consistent with these results, we show that intestinal AGT activity is effectively abolished by administration of 100 mg/kg temozolomide, even in the absence of BeG. In contrast, pretreatment with BeG transiently abolished the apoptotic response to the methylating prodrug dacarbazine. Activation of dacarbazine to its reactive intermediate has previously been shown to be cytochrome P450 dependent and we show here that pretreatment of mice with the cytochrome P450 inhibitor metyrapone also inhibits dacarbazine-induced apoptosis. Thus BeG increases neither the prevalence of apoptosis nor mutation frequency in the murine small intestine, but is capable of inhibiting P450-dependent prodrug activation. The positive implication from this study is that BeG treatment may not exacerbate the toxic and mutagenic effects of methylating agents within normal cells, although it may engender other adverse reactions through the suppression of cytochrome P450-dependent processes.

Creating effective social marketing: let your customer be your guide.

PIP: Social marketing uses commercial marketing techniques to change behaviors that benefit individuals or society in general. Unlike conventional marketing, which seeks to sell products or services, social marketing aims to promote voluntary behavior change. Some examples of behaviors that have changed due to social marketing are: using seat belts, wearing bike helmets, child immunizations, and smoking cessation. Although good social marketing campaigns use the same techniques as that of commercial marketers, by letting the customer be the guide for all major decisions, it is not primarily advertising and is not about top-down planning and decisions. Instead, it is about having a consumer orientation, which means understanding the target audience very well. An effective social marketer must be committed to ongoing communication with the audience in order to create programs, products, or practice that enable them to make the changes desired.^ieng

Potent induction of focused Th1-type cellular and humoral immune responses by RTS,S/SBAS2, a recombinant Plasmodium falciparum malaria vaccine.

The RTS,S/SBAS2 vaccine confers sterile protection against Plasmodium falciparum sporozoite challenge. The mechanisms underlying this are of great interest, yet little is known about the immune effector mechanisms induced by this vaccine. The immune responses induced by RTS,S/SBAS2 were characterized in 10 malaria-naive volunteers. Several epitopes in the circumsporozoite protein (CSP) were identified as targets of cultured interferon (IFN)-gamma-secreting CD4+ T cells. RTS,S-specific IFN-gamma-secreting effector T cells were induced in 8 subjects; this ex vivo response mapped to a single peptide in Th2R. CSP-specific CD8+ cytotoxic T lymphocytes were not detected. RTS, S-specific IFN-gamma production was universal, whereas interleukin-4 and -5 production was rare. RTS,S-specific lymphoproliferative responses and antibodies to CSP were strongly induced in all volunteers. Responses waned with time but were boostable. Thus, RTS, S/SBAS2 is a potent inducer of Th1-type cellular and humoral immunity. These results highlight possible immune mechanisms of protection and have important implications for vaccine design in general.

Homing of mononuclear cells from iliac lymph nodes to the genital and rectal mucosa in non-human primates.

The route of immunization may affect the type of immunity that is induced. The objectives of this investigation were to establish in the non-human primate if the internal iliac lymph nodes (LN) function as an inductive site of immunity from which mononuclear cells home to the rectal and cervico-vaginal mucosa. Rhesus macaques were immunized with simian immunodeficiency virus (SIV) core antigen p27 in the proximity of the iliac lymph nodes, and compared with the intramuscular (i.m.) (deltoid or gluteal), and axillary LN routes of immunization. The macaques were then challenged rectally or vaginally by a particulate SIVp27 antigen which was applied to the mucosal surface. The tracking dye PKH26 was injected near the immunizing LN or i.m. site and a week later the mucosal and lymphoid tissues were examined at autopsy. Preferential homing of PKH26-labeled cells from the internal iliac LN to the rectal and vaginal mucosa was demonstrated by flow cytometry after targeted iliac LN (TILN) but not after intramuscular (deltoid) or axillary LN immunization. Homing of the subsets of cells revealed that labeled CD4, CD8 and B cells, as well as monocytes were found in the rectum, colon, vagina or cervix. The results of this investigation shows that the route of immunization may affect regional mucosal immunity. Furthermore, the internal iliac LN may function as an inductive immunological site from which CD4, CD8 and B cells may home preferentially to the rectal, cervical and vaginal mucosa, as well as to the related regional but not the unrelated distal LN.

1,25-Dihydroxyvitamin D3 induces nitric oxide synthase and suppresses growth of Mycobacterium tuberculosis in a human macrophage-like cell line.

Inducible synthesis of nitric oxide (NO) by macrophages is an important mechanism of the host defense against intracellular infection in mice, but the evidence for significant levels of inducible NO production by human macrophages is controversial. Here we report that the human promyelocytic cell line HL-60, when differentiated to a macrophage-like phenotype, acquires the ability to produce substantial amounts of NO on stimulation with LPS or 1, 25-dihydroxyvitamin D3 (1,25-D3) in the absence of activating factors such as gamma interferon. Expression of the inducible nitric oxide synthase (NOS2) was confirmed by sequencing of the reverse transcription-PCR product from stimulated HL-60 cells. Kinetic studies after lipopolysaccharide stimulation show that NOS2 mRNA levels rise within 3 to 6 h, that conversion of [14C]arginine to [14C]citrulline is maximal at 5 to 6 days, and that levels of reactive nitrogen intermediates stabilize at around 20 microM at 7 to 8 days. We find that 1,25-D3 acts to suppress the growth of Mycobacterium tuberculosis in these cells and that this effect is inhibited by NG-monomethyl-L-arginine, suggesting that vitamin D-induced NO production may play a role in the host defense against human tuberculosis.