Retroviral-mediated gene transfer restores IL-12 and IL-23 signaling pathways in T cells from IL-12 receptor beta1-deficient patients.

Genetic deficiency of human IL-12 receptor beta1 chain (IL-12Rbeta1) results in increased vulnerability to weakly pathogenic strains of Mycobacteria and Salmonella. This phenotype results from the combined lack of IL-12 and IL-23 signaling as both cytokine receptors share IL-12Rbeta1. Such infections can be treated by administration of antibiotics and IFN-gamma; however, patients can succumb to infections despite these treatments. Reversion of patients' susceptibility by corrective gene transfer could prevent the infectious episodes, thus providing a beneficial alternative. We therefore evaluated the feasibility of retroviral-mediated gene correction of T cells obtained from patients carrying "null" mutations of IL-12Rbeta1. Transduction of the IL-12Rbeta1 cDNA restored the expression of IL-12Rbeta1 and resulted in the reconstitution of a functional IL-12 signaling pathway, as demonstrated by STAT4 phosphorylation and IFN-gamma production. IFN-gamma production in response to IL-23 was also corrected after gene transfer. These results indicate that the biological defects of T cells from patients carrying IL-12Rbeta1 deficiency can be corrected by gene transfer and form the basis for further development of gene therapy for this disease.     

Inherited interleukin 12 deficiency in a child with bacille Calmette-Guérin and Salmonella enteritidis disseminated infection.

Interferon-gamma receptor ligand-binding chain (IFN-gammaR1) or signaling chain (IFN-gammaR2) deficiency, like interleukin 12 receptor beta1 chain (IL-12Rbeta1) deficiency, predispose to severe infections due to poorly virulent mycobacteria and salmonella. A child with bacille Calmette-Guérin and Salmonella enteritidis infection was investigated. Mutations in the genes for IFN-gammaR1, IFN-gammaR2, IL-12Rbeta1, and other molecules implicated in IL-12- or IFN-gamma-mediated immunity were sought. A large homozygous deletion within the IL-12 p40 subunit gene was found, precluding expression of functional IL-12 p70 cytokine by activated dendritic cells and phagocytes. As a result, IFN-gamma production by lymphocytes was markedly impaired. This is the first discovered human disease resulting from a cytokine gene defect. It suggests that IL-12 is essential to and appears specific for protective immunity to intracellular bacteria such as mycobacteria and salmonella.     

Selective predisposition to bacterial infections in IRAK-4-deficient children: IRAK-4-dependent TLRs are otherwise redundant in protective immunity

Human interleukin (IL) 1 receptor–associated kinase 4 (IRAK-4) deficiency is a recently discovered primary immunodeficiency that impairs Toll/IL-1R immunity, except for the Toll-like receptor (TLR) 3– and TLR4–interferon (IFN)-a/b pathways. The clinical and immunological phenotype remains largely unknown. We diagnosed up to 28 patients with IRAK-4 deficiency, tested blood TLR responses for individual leukocyte subsets, and TLR responses for multiple cytokines. The patients' peripheral blood mononuclear cells (PBMCs) did not induce the 11 non-IFN cytokines tested upon activation with TLR agonists other than the nonspecific TLR3 agonist poly(I:C). The patients' individual cell subsets from both myeloid (granulocytes, monocytes, monocyte-derived dendritic cells [MDDCs], myeloid DCs [MDCs], and plasmacytoid DCs) and lymphoid (B, T, and NK cells) lineages did not respond to the TLR agonists that stimulated control cells, with the exception of residual responses to poly(I:C) and lipopolysaccharide in MDCs and MDDCs. Most patients (22 out of 28; 79%) suffered from invasive pneumococcal disease, which was often recurrent (13 out of 22; 59%). Other infections were rare, with the exception of severe staphylococcal disease (9 out of 28; 32%). Almost half of the patients died (12 out of 28; 43%). No death and no invasive infection occurred in patients older than 8 and 14 yr, respectively. The IRAK-4–dependent TLRs and IL-1Rs are therefore vital for childhood immunity to pyogenic bacteria, particularly Streptococcus pneumoniae. Conversely, IRAK-4–dependent human TLRs appear to play a redundant role in protective immunity to most infections, at most limited to childhood immunity to some pyogenic bacteria.     

Infections in the immunocompromised rheumatologic patient

Immunocompromised patients with rheumatic diseases have an increased risk of infections. A major risk factor for infection seems to be the immunosuppressive therapy used. Newer therapies for RA may lead to increased rates of infection by opportunistic pathogens such as Mycobacteria tuberculosis. Because disease manifestation may mimic signs and symptoms of infection, prompt diagnosis may be difficult. Familiarity with the likely infections and their causes should aid in obtaining the appropriate culture specimens.     

Glutathione deficiency in type 2 diabetes impairs cytokine responses and control of intracellular bacteria

Individuals with type 2 diabetes are at increased risk of acquiring melioidosis, a disease caused by Burkholderia pseudomallei infection. Although up to half of melioidosis patients have underlying diabetes, the mechanisms involved in this increased susceptibility are unknown. We found that B. pseudomallei-infected PBMCs from diabetic patients were impaired in IL-12p70 production, which resulted in decreased IFN-γ induction and poor bacterial killing. The defect was specific to the IL-12-IFN-γ axis. Defective IL-12 production was also observed during Mycobacterium tuberculosis infection, in which diabetes is likewise known to be a strong risk factor. In contrast, IL-12 production in diabetic cells was not affected upon Salmonella enterica infection or in response to TLR2, -3, -4, and -5 ligands. Poor IL-12 production correlated with a deficiency in intracellular reduced glutathione (GSH) concentrations in diabetic patients. Addition of GSH or N-acetylcysteine to PBMCs selectively restored IL-12 and IFN-γ production and improved bacterial killing. Furthermore, the depletion of GSH in mice led to increased susceptibility to melioidosis, reduced production of IL-12p70, and poorer disease outcome. Our data thus establish a link between GSH deficiency in diabetes and increased susceptibility to melioidosis that may open up new therapeutic avenues to protect diabetic patients against some intracellular bacterial pathogens.     

Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity

Autosomal dominant TLR3 deficiency has been identified as a genetic etiology of childhood herpes simplex virus 1 (HSV-1) encephalitis (HSE). This defect is partial, as it results in impaired, but not abolished induction of IFN-β and -λ in fibroblasts in response to TLR3 stimulation. The apparently normal resistance of these patients to other infections, viral illnesses in particular, may thus result from residual TLR3 responses. We report here an autosomal recessive form of complete TLR3 deficiency in a young man who developed HSE in childhood but remained normally resistant to other infections. This patient is compound heterozygous for two loss-of-function TLR3 alleles, resulting in an absence of response to TLR3 activation by polyinosinic-polycytidylic acid (poly(I:C)) and related agonists in his fibroblasts. Moreover, upon infection of the patient's fibroblasts with HSV-1, the impairment of IFN-β and -λ production resulted in high levels of viral replication and cell death. In contrast, the patient's peripheral blood mononuclear cells responded normally to poly(I:C) and to all viruses tested, including HSV-1. Consistently, various TLR3-deficient leukocytes from the patient, including CD14(+) and/or CD16(+) monocytes, plasmacytoid dendritic cells, and in vitro derived monocyte-derived macrophages, responded normally to both poly(I:C) and HSV-1, with the induction of antiviral IFN production. These findings identify a new genetic etiology for childhood HSE, indicating that TLR3-mediated immunity is essential for protective immunity to HSV-1 in the central nervous system (CNS) during primary infection in childhood, in at least some patients. They also indicate that human TLR3 is largely redundant for responses to double-stranded RNA and HSV-1 in various leukocytes, probably accounting for the redundancy of TLR3 for host defense against viruses, including HSV-1, outside the CNS.     

BCG turns 100: its nontraditional uses against viruses,cancer, and immunologic diseases

First administered to a human subject as a tuberculosis (TB) vaccine on July 18, 1921, Bacillus Calmette-Guérin (BCG) has a long history of use for the prevention of TB and later the immunotherapy of bladder cancer. For TB prevention, BCG is given to infants born globally across over 180 countries and has been in use since the late 1920s. With about 352 million BCG doses procured annually and tens of billions of doses having been administered over the past century, it is estimated to be the most widely used vaccine in human history. While its roles for TB prevention and bladder cancer immunotherapy are widely appreciated, over the past century, BCG has been also studied for nontraditional purposes, which include (a) prevention of viral infections and nontuberculous mycobacterial infections, (b) cancer immunotherapy aside from bladder cancer, and (c) immunologic diseases, including multiple sclerosis, type 1 diabetes, and atopic diseases. The basis for these heterologous effects lies in the ability of BCG to alter immunologic set points via heterologous T cell immunity, as well as epigenetic and metabolomic changes in innate immune cells, a process called “trained immunity.” In this Review, we provide an overview of what is known regarding the trained immunity mechanism of heterologous protection, and we describe the current knowledge base for these nontraditional uses of BCG.     

The IL-12Rbeta2 gene functions as a tumor suppressor in human B cell malignancies

The IL-12Rbeta2 gene is expressed in human mature B cell subsets but not in transformed B cell lines. Silencing of this gene may be advantageous to neoplastic B cells. Our objective was to investigate the mechanism(s) and the functional consequence(s) of IL-12Rbeta2 gene silencing in primary B cell tumors and transformed B cell lines. Purified tumor cells from 41 patients with different chronic B cell lymphoproliferative disorders, representing the counterparts of the major mature human B cell subsets, tested negative for IL-12Rbeta2 gene expression. Hypermethylation of a CpG island in the noncoding exon 1 was associated with silencing of this gene in malignant B cells. Treatment with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine restored IL-12Rbeta2 mRNA expression in primary neoplastic B cells that underwent apoptosis following exposure to human recombinant IL-12 (hrIL-12). hrIL-12 inhibited proliferation and increased the apoptotic rate of IL-12Rbeta2-transfected B cell lines in vitro. Finally, hrIL-12 strongly reduced the tumorigenicity of IL-12Rbeta2-transfected Burkitt lymphoma RAJI cells in SCID-NOD mice through antiproliferative and proapoptotic effects, coupled with neoangiogenesis inhibition related to human IFN-gamma-independent induction of hMig/CXCL9. The IL-12Rbeta2 gene acts as tumor suppressor in chronic B cell malignancies, and IL-12 exerts direct antitumor effects on IL-12Rbeta2-expressing neoplastic B cells.     

Infection of Mice with Mycobacterium bovis–Bacillus Calmette-Guérin (BCG) Suppresses Allergen-induced Airway Eosinophilia

It has been proposed that the increase in prevalence and severity of atopic disorders inversely correlates with exposure to infectious diseases such as tuberculosis. We have investigated this issue by combining an intranasal Mycobacterium bovis–Bacillus Calmette-Guérin (BCG) infection with a murine model of allergen, (ovalbumin [OVA]) induced airway eosinophilia. BCG infection either 4 or 12 wk before allergen airway challenge resulted in a 90–95 and 60–70% reduction in eosinophilia within the lungs, respectively, compared to uninfected controls. The inhibition of airway eosinophilia correlated with a reduced level of IL-5 production by T cells from the lymph node draining the site of OVA challenge. Interestingly, BCG infection of the lung had no effect on IgG1 and IgE OVA-specific serum immunoglobulin or blood eosinophil levels. Furthermore, BCG-induced inhibition of airway eosinophilia was strongly reduced in interferon (IFN)-γ receptor–deficient mice and could be partially reversed by intranasal IL-5 application. Intranasal BCG infections could also reduce the degree of lung eosinophilia and IL-5 produced by T cells after Nippostrongylus brasiliensis infection. Taken together, our data suggest that IFN-γ produced during the T helper cell (Th)1 immune response against BCG suppresses the development of local inflammatory Th2 responses in the lung. Most importantly, this inhibition did not extend to the systemic immunoglobulin response against OVA. Our data support the view that mycobacterial infections have the potential to suppress the development of atopic disorders in humans.     

Polarized type-1 dendritic cells (DC1) producing high levels of IL-12 family members rescue patient TH1-type antimelanoma CD4+ T cell responses in vitro

Deficiencies in TH1-type immunity in patients with cancer may facilitate tumor progression and limit the effectiveness of current immunotherapy approaches. We hypothesized that Type-1 polarized dendritic cells (DC1) might be able to recondition patient antitumor CD4+ T cell responses toward the TH1-type in vitro. Although DC1 have been previously demonstrated to prime TH1 responses from naive CD4+ T cells, their impact on antigen-experienced TH responses remains unknown. We confirmed our own earlier observations that patient CD4+ T cell reactivity against melanoma-associated antigens (MAA) was weaker and less Type-1-polarized than their corresponding antiviral responses. Stimulation of patient CD4 T cells with peptide-pulsed DC1 (producing multiple IL-12 family member cytokines, including IL-12p70, IL-23, and IL-27) promoted robust TH1-type, epitope-specific T cell responses. Addition of exogenous IL-12 family member cytokines alone, or in combination, to nonpolarized DC was insufficient to equate to the benefits associated with DC1-based stimulation; however, IL-27 and IL-12p70 blockade neutralized the ability of DC1 cells to enhance TH1-type antitumor immunity in vitro. Notably, DC1-based stimulation seemed capable of "revitalizing" defective TH1-type responses within the CD45RO+ subset of antigen-experienced CD4+ T cells in melanoma patients. In addition to promoting elevated levels of IFN-gamma from responder CD4+ T cells, DC1-based stimulation also led to increased levels of IL-12Rbeta2 and t-bet expression by TH cells. These results suggest that preexisting CD4+ T cell immunity to cancer is not relegated to Type-1 insufficiency and may be corrected via the application of DC1-based vaccination protocols.     

Tuberculosis and atypical mycobacterioses in HIV infection. Results from the Bonn Center 1985 to 1989

485 HIV-positive patients have been treated at our institution in Bonn during 1985 to 1989. Mycobacterial infections occurred in twelve (2.5%) HIV-positive patients. Of 166 AIDS-manifestations according to CDC, eleven (6.6%) were mycobacterial infections. There occurred one case of miliary tuberculosis, six cases of extrapulmonary, one of disseminated tuberculosis and four cases of atypical mycobacteriosis. Mycobacteriosis other than tuberculosis (MOTT) were caused three times by Mycobacterium kansasii and once by Mycobacterium scrofulaceum. Tuberculosis was seen less often in haemophiliacs. Disseminated tuberculosis and atypical mycobacteriosis developed in late stages of HIV-infection with underlying severe immunodeficiency. The lung was the main target organ of tuberculosis. MOTT most often affected the gastrointestinal tract additionally. Noninvasive materials, first of all sputum and gastric acid, were reliably diagnostic but available with delay in particular cases. In those cases histologic studies proved helpful. Application of five-fold regimen (INH, RMP, EMB, PZA and SM) always succeeded in negative cultures in a mean of 15 days in all cases of tuberculosis. Two cases of atypical mycobacteriosis with Mycobacterium kansasii were treated with a five-fold regimen (one case with ciprofloxacin additionally) and culture-negative after six resp. 28 weeks of therapy.     

Early interleukin 12 production by macrophages in response to mycobacterial infection depends on interferon gamma and tumor necrosis factor alpha

Interleukin 12 (IL-12) produced by macrophages immediately after infection is considered essential for activation of a protective immune response against intracellular pathogens. In the murine Mycobacterium bovis Bacillus Calmette-Guerin (BCG) model we assessed whether early IL- 12 production by macrophages depends on other cytokines. In vitro, murine bone marrow-derived macrophages produced IL-12 after infection with viable M. bovis BCG or stimulation with LPS, however, priming with recombinant interferon gamma (rIFN-gamma) was necessary. In addition, IL-12 production by these macrophages was blocked by specific anti- tumor necrosis factor alpha (TNF-alpha) antiserum. Macrophages from gene deletion mutant mice lacking either the IFN-gamma receptor or the TNF receptor 1 (p55) failed to produce IL-12 in vitro after stimulation with rIFN-gamma and mycobacterial infection. In vivo, IL-12 production was induced in spleens of immunocompetent mice early during M. bovis BCG infection but not in those of mutant mice lacking the receptors for IFN-gamma or TNF. Our results show that IL-12 production by macrophages in response to mycobacterial infection depends on IFN-gamma and TNF. Hence, IL-12 is not the first cytokine produced in mycobacterial infections.     

The etiological structure of acute intestinal infections]

The share of various etiologic forms of acute intestinal infections, diagnosed by bacteriologic methods, is presented. The share of gastroenterocolitis induced by opportunistic microflora makes up 35.6%, that of dysentery 25.6%, salmonellosis 18.5%; mixed infection (dysentery + salmonellosis) is diagnosed in 7% of cases with acute intestinal infections. The principal representative of opportunistic microflora isolated from patients with acute intestinal infections is the Klebsiella genus (40.6%), whereas in the reference group Citrobacter, Morganella, and Klebsiella detection rates are approximately the same (27.0-18.3%). Opportunistic microorganisms in titers under 10(6) are isolated from normal subjects 5 times more frequently than from the patients, this indicating the diagnostic value of this level of feces contamination with opportunistic microflora.     

T helper type 2 cell differentiation occurs in the presence of interleukin 12 receptor beta2 chain expression and signaling

The differentiation of CD4(+) T cells into T helper type 1 (Th1) cells is driven by interleukin (IL)-12 through the IL-12 receptor beta2 (IL-12Rbeta2) chain, whereas differentiation into Th2 cells is driven by IL-4, which downregulates IL-12Rbeta2 chain. We reexamined such differentiation using IL-12Rbeta2 chain transgenic mice. We found that CD4(+) T cells from such mice were able to differentiate into Th2 cells when primed with IL-4 or IL-4 plus IL-12. In the latter case, the presence of IL-4 suppressed interferon (IFN)-gamma production 10-100-fold compared with cells cultured in IL-12 alone. Finally, in studies of the ability of IL-12 to convert Th2 cells bearing a competent IL-12R to the Th1 cells, we showed that: (a) T cells bearing the IL-12Rbeta2 chain transgene and primed under Th2 conditions could not be converted to Th1 cells by repeated restimulation under Th1 conditions; and (b) established Th2 clones transfected with the IL-12Rbeta2 chain construct continued to produce IL-4 when cultured with IL-12. These studies show that IL-4-driven Th2 differentiation can occur in the presence of persistent IL-12 signaling and that IL-4 inhibits IFN-gamma production under these circumstances. They also show that established Th2 cells cannot be converted to Th1 cells via IL-12 signaling.     

Time course changes in cytokines content and gas composition in blood of patients with salmonellosis and acute shigellosis

AIM: To study changes in serum concentrations of interleukines (IL) and tumor necrosis factor alpha (TNFa) in the course of acute moderate and severe shigellosis and salmonellosis; to elicit their correlation with disorders of gas and electrolyte blood composition and acid-base balance (ABB). MATERIAL AND METHODS: A total of 39 patients with salmonellosis and 32 patients with acute shigellosis admitted to infectious hospital N 2 entered the study. The following parameters were assessed: serum concentrations of IL-1, IL-IO and TNFa; ABB, gas and electrolyte blood composition; leucocytic intoxication index (LII). The tests were made on the disease day 2-3 and 6-7 (in severe salmonellosis on day 10-11). RESULTS: In moderate salmonellesis and acute shigellosis the level of proinflammatory cytokines (IL-1 and TNFa) diminished while in severe acute intestinal infection their concentration was high reflecting imbalance of immune response. Content of IL-10 depends on etiology and severity of the course of infectious process - the highest IL-10 concentrations were found in patients with severe salmonellosis on the disease day 10-11. A direct correlation was confirmed between IL-1, TNFa and LII in the disease onset in all patients with acute intestinal infections. CONCLUSION: An important role of IL-1, IL-10 and TNFa in pathogenesis of bacterial intestinal infections is confirmed. A correlation exists between blood gas composition and concentration of cytokines.     

In vitro activity of roxithromycin against the Mycobacterium tuberculosis complex.

Roxithromycin has recently been shown to possess significant in vitro activity against a variety of atypical mycobacteria such as the M. avium complex, M. scrofulaceum, M. szulgai, M. malmoense, M. xenopi, M. marinum, and M. kansasii and rare pathogens like M. chelonei and M. fortuitum. In the present investigation, screening of its in vitro activity was further extended by testing it against 34 strains belonging to the M. tuberculosis complex (including M. tuberculosis, M. africanum, M. bovis, and M. bovis BCG). The MICs were determined by the radiometric BACTEC 460-TB methodology at pHs of both 6.8 and 7.4, as well as with 7H10 agar medium by the 1% proportion method. With the exception of M. bovis BCG (MIC ranges, 0.5 to 4 micrograms/ml at pH 6.8 and 0.25 to 2 micrograms/ml at pH 7.4), MICs for all of the isolates were significantly greater (MIC ranges, 32 to > 64 micrograms/ml at pH 6.8 and 16 to > 32 micrograms/ml at pH 7.4) than those reported previously for atypical mycobacteria. Roxithromycin MICs of 64 or > 64 micrograms/ml for all of the M. tuberculosis isolates screened were found by the 7H10 agar medium method. Roxithromycin, however, showed a pH-dependent bactericidal effect against M. tuberculosis because the drug was relatively more active when it was used at pH 7.4 than when it was used at pH 6.8. We conclude that roxithromycin per se is not a drug of choice for the treatment of M. tuberculosis infection or disease; however, considering its pharmacokinetics, eventual anti-tubercle bacillus activity in an in vivo system cannot yet be excluded. We suggest that the use of roxithromycin in chemoprophylactic regimens for the prevention of opportunistic infections (including M. avium complex infections) in patients with AIDS should be carefully monitored, and patients should be enrolled in such a regimen only after it has been excluded that the patient das an underlying infection of disease caused by M. tuberculosis.     

缺铁性贫血合并反复呼吸道感染患儿细胞免疫功能观察

目的：测定６３例缺铁性贫血（ＩＤＡ）合并反复呼吸道感染（ＲＲＩ）患儿外周血白细胞介素２（ＩＬ－２）活性、血清可溶性白细胞介素２受体（ｓＩＬ－２Ｒ）水平和Ｔ淋巴细胞亚群，并分析它们之间的相关性。方法：ＩＬ－２活性和ｓＩＬ－２Ｒ水平测定分别采用ＭＴＴ法和双抗体夹心法，Ｔ淋巴细胞亚群测定采用ＡＰＡＡＰ法。结果：ＩＬ－２活性，ＣＤ３＋、ＣＤ４＋细胞百分率和ＣＤ４＋／ＣＤ８＋细胞比值均显著低于对照组（Ｐ均＜０．０１），而ｓＩＬ－２Ｒ水平显著高于对照组（Ｐ＜０．０１），ＣＤ８＋细胞百分率无明显变化。结论：ＩＤＡ合并ＲＲＩ患儿细胞免疫功能低下且紊乱。