Proteomic analysis reveals critical molecular mechanisms involved in the macrophage anti-spinal tuberculosis process.

Tuberculosis infection activates the autoimmune system. However, the role of host-pathogen interactions involved in Mycobacterium tuberculosis infection is unclear. In this study, we analyzed 6 spinal tuberculosis tissues and 6 herniated disc tissues by using liquid chromatography-tandem mass spectrometry coupled with tandem mass spectrometry, and immunohistochemical staining was performed for validating the results. We identified 42 differential immune-related proteins and 3 hub genes that are primarily localised in the tertiary granule and involved in biological processes such as cellular response to the presence of cadmium ions, regulation of ion transmembrane transport, transmembrane transport, and inflammatory responses. Genes encoding cytochrome B-245 beta chain (CYBB), matrix metallopeptidase 9 (MMP9), and C-X-C motif chemokine ligand 10 (CXCL10) were identified as the hub genes that exhibited anti-tuberculosis activity and were responsible for macrophage resistance against M. tuberculosis. In conclusion, CYBB, MMP9, and CXCL10 resist M. tuberculosis infection through chemotaxis and macrophage activation. Our results indicate that CYBB, MMP9, and CXCL10 could be considered as molecular targets for spinal tuberculosis treatment, which may significantly improve patients' quality of life and prognosis.

Elevated expression of lncRNA SNHG15 in spinal tuberculosis: preliminary results.

OBJECTIVE: The incidence and disability rate of spinal tuberculosis is high. The role of the expression of lncRNA SNHG15 in spinal tuberculosis and related mechanisms remains unclear. PATIENTS AND METHODS: Spinal tuberculosis and normal control tissues were collected, and lncRNA SNHG15 level was analyzed by real-time PCR. Mouse RAW264.7 cells were cultured and divided into control group, tuberculin (PPD) group, si-SNHG15, and PPD+ si-SNHG15 group followed by analysis of lncRNA SNHG15 level, cell proliferation by MTT assay, formation of osteoclasts by TRAP staining, levels of interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) by ELISA, as well as expression of RANK and RANKL by Western blot. RESULTS: The lncRNA SNHG15 expression in spinal tuberculosis tissues was significantly increased compared with that in the control group (p < 0.05). The expression of lncRNA SNHG15 was increased in RAW264.7 cells in the PPD group with increased cell proliferation, TRAP-positive cells, IL-6 and TNF-α secretion, as well as elevated RANK and RANKL expression which were statistically different compared with the control group (p < 0.05). Transfection of lncRNA SNHG15 siRNA in the PPD model significantly inhibited the expression of lncRNA SNHG15, decreased cell proliferation, TRAP staining positive cells, IL-6 and TNF-α secretion, as well as reduced RANK and RANKL expression. Compared with the PPD group, the differences were statistically significant (p < 0.05). CONCLUSIONS: The expression of lncRNA SNHG15 was significantly increased in spinal tuberculosis tissues. The downregulation of lncRNA SNHG15 expression could inhibit the secretion of inflammatory cytokines by regulating the RANK/RANKL pathway, thereby regulating osteoclasts.

One-Stage Surgical Treatment for Consecutive Multisegment Thoracic Spinal Tuberculosis with Kyphosis by Posterior-Only Debridement, Interbody Fusion, and Instrumentation.

OBJECTIVE: To evaluate the clinical efficacy and feasibility of one-stage surgical treatment for consecutive multisegment thoracic spinal tuberculosis with kyphosis by posterior-only debridement, interbody fusion, and instrumentation. METHODS: Sixty-two patients who underwent posterior debridement, interbody fusion, and instrumentation were reviewed for radiographic fusion, region kyphosis, neurologic status, and clinical outcomes. Thoracic Cobb's angle and Frankel grading system were used to assess kyphosis and neurologic improvements, respectively. Operation time, blood loss, erythrocyte sedimentation rate, C-reactive protein, visual analogue scale score, and postoperative complications were recorded to evaluate efficacy and feasibility. RESULTS: The surgery duration was 234.5 ± 91.3 minutes, with blood loss of 761.3 ± 598.5 mL. The levels of erythrocyte sedimentation rate and C-reactive protein in all patients decreased gradually to normal within 3 months after the surgery. Kyphosis angle was corrected from 16.9 ± 10.9° preoperatively to 10.4 ± 6.3° postoperatively (P < 0.001, t = 5.2) and remained at 12.0 ± 6.6° at final follow-up (P < 0.001, t = 4.6). Twenty-seven patients obtained neurologic improvement by 1-3 grades. The average visual analogue scale score decreased from preoperative 3.7 ± 1.0 to postoperative 0.7 ± 0.9 (P < 0.001, t = 16.4), and then to 0.5 ± 0.3 at final follow-up (P < 0.001, t = 21.5). All patients achieved bony fusion. Recurrence of tuberculosis was not found in all patients. CONCLUSIONS: One-stage posterior surgery of debridement, interbody fusion and instrumentation could serve as an efficient way to cure patients with consecutive multisegment thoracic spinal tuberculosis.

Roles of monocyte chemotactic protein 1 and nuclear factor-κB in immune response to spinal tuberculosis in a New Zealand white rabbit model.

This study aimed to explore the roles of monocyte chemotactic protein 1 (MCP-1) and nuclear factor kappa B (NF-κB) in immune response to spinal tuberculosis in a New Zealand white rabbit model. Forty-eight New Zealand white rabbits were collected and divided into four groups: experimental group (n=30, spinal tuberculosis model was established), the sham group (n=15, sham operation was performed) and the blank group (n=3). The qRT-PCR assay and western blotting were applied to detect the mRNA and protein expressions of MCP-1 and NF-κB in peripheral blood. ELISA was used to measure serum levels of MCP-1, NF-κB, IFN-γ, IL-2, IL-4, and IL-10. Flow cytometry was adopted to assess the distributions of CD4+, CD8+ lymphocytes and CD4+ CD25+ Foxp3 lymphocyte subsets. Compared with the sham and blank groups, the mRNA and protein expressions of MCP-1 and NF-κB in the experimental group were significantly increased. The experimental group had lower serum levels of IL-2 and IFN-γ and higher serum level of IL-10 than the sham and blank groups. In comparison to the sham and blank groups, CD4+ T lymphocyte subsets percentage, CD4+/CD8+ ratio and CD4+ CD25+ Foxp3+ Tregs subsets accounting for CD4+ lymphocyte in the experimental group were lower, while percentage of CD8+ T lymphocyte subsets was higher. Our study provided evidence that higher expression of MCP-1 and NF-κB may be associated with decreased immune function of spinal tuberculosis, which can provide a new treatment direction for spinal tuberculosis.

Roles of monocyte chemotactic protein 1 and nuclear factor-kappaB in immune response to spinal tuberculosis in a New Zealand white rabbit model

This study aimed to explore the roles of monocyte chemotactic protein 1 (MCP-1) and nuclear factor kappa B (NF-κB) in immune response to spinal tuberculosis in a New Zealand white rabbit model. Forty-eight New Zealand white rabbits were collected and divided into four groups: experimental group (n=30, spinal tuberculosis model was established), the sham group (n=15, sham operation was performed) and the blank group (n=3). The qRT-PCR assay and western blotting were applied to detect the mRNA and protein expressions of MCP-1 and NF-κB in peripheral blood. ELISA was used to measure serum levels of MCP-1, NF-κB, IFN-γ, IL-2, IL-4, and IL-10. Flow cytometry was adopted to assess the distributions of CD4+, CD8+ lymphocytes and CD4+ CD25+ Foxp3 lymphocyte subsets. Compared with the sham and blank groups, the mRNA and protein expressions of MCP-1 and NF-κB in the experimental group were significantly increased. The experimental group had lower serum levels of IL-2 and IFN-γ and higher serum level of IL-10 than the sham and blank groups. In comparison to the sham and blank groups, CD4+ T lymphocyte subsets percentage, CD4+/CD8+ ratio and CD4+ CD25+ Foxp3+ Tregs subsets accounting for CD4+ lymphocyte in the experimental group were lower, while percentage of CD8+ T lymphocyte subsets was higher. Our study provided evidence that higher expression of MCP-1 and NF-κB may be associated with decreased immune function of spinal tuberculosis, which can provide a new treatment direction for spinal tuberculosis.

Immunotherapy for non-responders among patients of spinal tuberculosis.

BACKGROUND: Combined chemo- and immunotherapy are the major advancement in the treatment of tuberculosis. Immunotherapy supposedly increases cure rate while reducing the duration of treatment and tissue damage. Non-responders are those patients of tuberculosis who do not respond to anti-tubercular therapy (ATT) in the desired manner despite the mycobacteria showing sensitivity to the given drugs. The role of immunotherapy in the treatment of this particular subset of patients has been investigated scarcely. METHODS: The present study included a retrospective review of prospectively collected clinico-radiological data of 14 non-responder patients who were taking ATT for spinal tuberculosis for a mean duration of 10.3 months. An immunotherapeutic regime comprising of single intramuscular injection of vitamin D 600,000IU, 3 days course of oral albendazole 200mg daily, salmonella vaccine 0.5ml intramuscular and influenza vaccine 0.5ml intramuscular were added to ATT. The vaccines and the course of oral albendazole were repeated after a month. RESULTS: Before immunotherapy, seven patients were partially dependent while other seven were completely dependent on others for activities of daily living. All except one patient after treatment became independent till last follow-up (p value <0.01). Post immunotherapy, ATT was continued for mean duration of 4.9 months with mean follow-up of 22.4 months. All patients showed good clinical response within 2-6 weeks after the initiation of immunotherapy. CONCLUSIONS: The crux to success of the immunotherapy regime is its potential to restore the existing Th1 Th2 imbalance and to provide substitute to the anergic and dysfunctional immune cells.

Disseminated tuberculosis after pregnancy progressed to paradoxical response to the treatment: report of two cases.

BACKGROUND: Early postpartum women are more likely to develop tuberculosis than nonpregnant women mainly due to immune reconstitution after delivery. Paradoxical response (PR) during antituberculosis treatment also arises via recovery from immunosuppression. However, no study focused on PR during antituberculosis treatment in a postpartum patient has been reported. CASE PRESENTATION: We present two sequential cases (Patient 1: 26-year-old; Patient 2: 29-year-old) of postpartum tuberculosis with pulmonary and extrapulmonary lesions (Patient 1: peritonitis; Patient 2: psoas abscess secondary to spondylitis). Both cases progressed to PR (worsening of pre-existing lung infiltrations (Patients 1, 2) and new contralateral effusion (Patient 2)) in a relatively short time after initiation of treatment (Patient 1: 1 week; Patient 2: 3 weeks), suggesting that immune modulations during pregnancy and delivery may contribute to the pathogenesis of both disseminated tuberculosis and its PR. The pulmonary lesions and effusion of both cases gradually improved without change of chemotherapy regimen. CONCLUSION: Physicians should recognize PR in tuberculosis patients with postpartum and then evaluate treatment efficacy.

Pott's disease in Moroccan children: clinical features and investigation of the interleukin-12/interferon-γ pathway.

SETTING: Tuberculosis spondylodiscitis (TS), or Pott's disease, an extra-pulmonary form of tuberculosis (TB), is rare and difficult to diagnose in children. Some cases of severe TB in children were recently explained by inborn errors of immunity affecting the interleukin-12/interferon-gamma (IL-12/IFN-γ) axis. OBJECTIVE: To analyse clinical data on Moroccan children with TS, and to perform immunological and genetic explorations of the IL-12/IFN-γ axis. DESIGN: We studied nine children with TS diagnosed between 2012 and 2014. We investigated the IL-12/IFN-γ circuit by both whole-blood assays and sequencing of the coding regions of 14 core genes of this pathway. RESULTS: A diagnosis of TS was based on a combination of clinical, biological, histological and radiological data. QuantiFERON(®)-TB Gold In-Tube results were positive in 75% of patients. Whole-blood assays showed normal IL-12 and IFN-γ production in all but one patient, who displayed impaired decreased response to IL-12. No candidate disease-causing mutations were detected in the exonic regions of the 14 genes. CONCLUSIONS: TS diagnosis in children remains challenging, and is based largely on imaging. Further investigations of TS in children are required to determine the role of genetic defects in pathways that may or may not be related to the IL-12/IFN-γ axis.

TUBERCULOSIS INFECTION MIGHT INCREASE THE RISK OF INVASIVE CANDIDIASIS IN AN IMMUNOCOMPETENT PATIENT.

Deep Candida infections commonly occur in immunosuppressed patients. A rare case of a multiple deep organ infection with Candida albicans and spinal tuberculosis was reported in a healthy young man. The 19-year-old man complained of month-long fever and lower back pain. He also had a history of scalded mouth syndrome. Coinfection with Mycobacterium tuberculosis and Candida albicans was diagnosed using the culture of aspirates from different regions. Symptoms improved considerably after antifungal and antituberculous therapy. This case illustrates that infection with tuberculosis might impair the host's immune system and increase the risk of invasive candidiasis in an immunocompetent patient.

Tuberculosis ineection might increase the risk f invasive candidiasis in an immmunocompetent patient / Tuberculose pode aumentar o risco de candidíase invasiva em paciente imunocompetente

Deep Candida infections commonly occur in immunosuppressed patients. A rare case of a multiple deep organ infection with Candida albicans and spinal tuberculosis was reported in a healthy young man. The 19-year-old man complained of month-long fever and lower back pain. He also had a history of scalded mouth syndrome. Coinfection with Mycobacterium tuberculosis and Candida albicans was diagnosed using the culture of aspirates from different regions. Symptoms improved considerably after antifungal and antituberculous therapy. This case illustrates that infection with tuberculosis might impair the host's immune system and increase the risk of invasive candidiasis in an immunocompetent patient.

As infecções profundas por Candida ocorrem geralmente em pacientes imunossuprimidos. Relatamos caso raro de infecções profundas em múltiplos órgãos por Candida albicans e neuro tuberculose em homem jovem saudável. Um jovem de 19 anos de idade queixou-se de febre e lombalgia há um mês. Relatava ainda histórico de síndrome da boca escaldada. Foi diagnosticada co-infecção por Mycobacterium tuberculosis e Candida albicans em cultura do aspirado de diferentes regiões do organismo. Os sintomas melhoraram significativamente após a terapia antifúngica e antituberculosa. Este caso é apresentado para mostrar que a tuberculose pode prejudicar o sistema imune do hospedeiro e aumentar o risco de candidíase invasiva em paciente imunocompetente.

Monocyte chemoattractant protein-1 in spinal tuberculosis: -362G/C genetic variant and protein levels in Chinese patients.

The objective of the study is to explore the possible association of the monocyte chemoattractant protein (MCP)-1-362G/C genetic polymorphism and plasma levels of MCP-1 in patients with spinal tuberculosis (TB). The MCP-1-362G/C (rs2857656) polymorphism and blood levels of MCP-1 in patients with spinal TB and healthy subjects were evaluated and compared. Three hundred thirty-two patients and 336 healthy subjects were genotyped using polymerase chain reaction and Sanger DNA sequencing technology. MCP-1 plasma levels were measured by a solid-phase enzyme-linked immunosorbent assay. When comparisons were made between patients and controls, the frequency of the MCP-1-362*C minor allele (55.4% versus 47.5%, P = 0.004, odds ratio [OR] = 1.376, 95% confidence interval [CI]: 1.109-1.706) and the carriers of the MCP-1-362*C allele (80.7% versus 71.4%, P = 0.005, OR = 1. 657, 95% CI: 1.167-2.403) were over-represented in patients. The mean MCP-1 plasma level in spinal TB patients was significantly higher than in controls (154.44 ± 68.81 pg/mL versus 36.69 ± 21.71 pg/mL, t = -5.85, P < 0.001). The patients with the CC genotype had the highest MCP-1 level (150.63 ± 73.89 pg/mL), followed by those with the GC genotype (108.63 ± 52.09 pg/mL, t = 2.351, P = 0.022) and GG (91.29 ± 54.31 pg/mL, t = 3.091, P = 0.003) homozygotes. We report the association of the -362G/C genetic polymorphism and increased plasma levels of MCP-1 in patients with spinal TB and nominate the -362*C minor allele as a risk factor for spinal TB in the Chinese population.

Mycobacterium tuberculosis escapes from the phagosomes of infected human osteoclasts reprograms osteoclast development via dysregulation of cytokines and chemokines.

Spinal tuberculosis is a condition characterized by massive resorption of the spinal vertebrae due to the infection with Mycobacterium tuberculosis (Mtb). However, the pathogenesis of spinal tuberculosis has not been established because it was almost completely eradicated by the establishment of antibiotic treatment in the mid-20th century. In this study, we investigated the inflammatory responses of human multinucleated osteoclasts infected with virulent Mtb strain. We found that the intracellular Mtb infection of multinuclear osteoclasts resulted in the rapid growth of Mtb and an osteolytic response, rather than inflammation. In response to Mtb infection, the mononuclear osteoclast precursors produced proinflammatory cytokines including tumor necrosis factor (TNF)-α, an intrinsic characteristic they share with macrophages. In contrast, highly fused multinucleated osteoclasts incapacitated the production of these cytokines. Instead, the intracellular Mtb inside multinuclear osteoclasts escaped from the endosome/phagosome, leading to a different pattern of osteoclast activation, with the production of chemokines such as CCL5, CCL17, CCL20, CCL22, CCL24, and CCL25. Moreover, intracellular infection with an avirulent Mtb strain resulted in diminished production of these chemokines. These findings indicate that intracellular Mtb infection in multinuclear osteoclasts reprograms osteoclast development via the dysregulation of cytokines and chemokines.

Enzyme-linked immunospot assay response to recombinant CFP-10/ESAT-6 fusion protein among patients with spinal tuberculosis: implications for diagnosis and monitoring of surgical therapy.

OBJECTIVE: This study aimed to assess the performance of a laboratory-developed recombinant CFP-10/ESAT-6 fusion protein (rCFP-10/ESAT-6)-based enzyme-linked immunospot (ELISPOT) assay for the diagnosis of spinal tuberculosis (TB) in China, and to evaluate the value of the ELISPOT assay for monitoring the efficacy of surgical treatment. METHODS: In the first part of the study, a total of 78 participants were consecutively recruited for ELISPOT using rCFP-10/ESAT-6 as a stimulus. The cutoff value for ELISPOT positivity was based on the results of receiver operating characteristic curve analysis. In the second part, this approach was evaluated in a prospective study including 102 patients with suspected spinal TB. Data on clinical characteristics of the patients and conventional laboratory results were collected, and blood samples were obtained for ELISPOT using rCFP-10/ESAT-6 as a stimulus. RESULTS: Among the 102 patients with suspected spinal TB, 11 were excluded from the study. Twenty-three patients (25.2%) had culture-confirmed TB and 29 (31.9%) patients had probable TB. Among the spinal TB patients, the ELISPOT had a sensitivity of 82.7%, compared to a sensitivity of 61.5% for the purified protein derivative (PPD) skin test. The specificity was 87.2% for ELISPOT and 46.2% for the PPD skin test among 39 subjects with non-TB disease. The number of spot-forming cells and/or the positive rate of the ELISPOT assay were associated with aging, emaciation, and paravertebral abscess. The number of subjects with responses to rCFP-10/ESAT-6 slightly decreased after surgical treatment in spinal TB patients. CONCLUSIONS: A laboratory-developed rCFP-10/ESAT-6 ELISPOT assay is a useful adjunct to current tests for the diagnosis of spinal TB.

Immunohistological characterization of spinal TB granulomas from HIV-negative and -positive patients.

Tuberculosis (TB) is mainly a disease of the lungs, but Mycobacterium tuberculosis (Mtb) can establish infection in virtually any organ in the body. Rising rates of extrapulmonary (EP) TB have been largely associated with the HIV epidemic, as patients co-infected with HIV show a four-fold higher risk of EPTB. Spinal TB (Pott's Disease), one of the most debilitating extrapulmonary forms of disease, is difficult to diagnose and can cause deformity and/or neurological deficits. This study examined the histopathology and distribution of immune cells within spinal TB lesions and the impact of HIV on pathogenesis. The overall structure of the spinal granulomas resembled that seen in lung lesions from patients with pulmonary TB. Evidence of efficient macrophage activation and differentiation were detectable within organized structures in the spinal tissue, irrespective of HIV status. Interestingly, the granulomatous architecture and macroscopic features were similar in all samples examined, despite a reversal in the ratio of infiltrating CD4 to CD8 T cells in the lesions from HIV-infected patients. This study provides a foundation to understand the mechanism of tissue destruction and disease progression in Spinal TB, enabling the future development of novel therapeutic strategies and diagnostic approaches for this devastating disease.

Microbiological and immunological diagnosis of tuberculous spondylodiscitis.

BACKGROUND: Tuberculous spondylodiscitis is one the many manifestations of active tuberculosis (TB) and can result following primary infection or, more frequently, from reactivation of active TB in subjects with latent TB. Definitive diagnosis of tuberculous spondylodiscitis requires the identification of Mycobacterium tuberculosis in the biological sample following microbiological analysis. AIM: To summarize the recent advancement in the diagnosis of TB, focusing on classical and molecular microbiological procedures, providing an overview on the recent advancements in the understanding of TB pathogenesis and their implications for the immunological diagnosis MATERIALS AND METHODS: Isolation in culture of the bacilli and detection using molecular tools are the gold standards, though sensitivity of these assays is significantly lower compared to what observed for pulmonary TB, making diagnosis of spinal TB challenging. RESULTS: The use of the interferon-gamma release assays (IGRAs) for the immunological diagnosis of TB infection could be of help and shall precede the invasive techniques, such as biopsy or surgery, required to obtain the biological sample. IGRAs measure the presence of effector T cells in the blood that can readily respond to an antigenic stimuli by secreting cytokines, and that are an indication of the presence of the bacilli in vivo. IGRAs are more sensitive and specific than the intradermic reaction of Mantoux, though both these immunological tests cannot distinguish between latent TB infection and active TB. CONCLUSIONS: A modern diagnosis of TB spondylodiscitis should rely on the use of microbiological and immunological assays and the latter could potentially be of great help in monitoring therapy effectiveness.

An atypical localisation of tuberculosis infection in patients undergoing haemodialysis: a case report.

Spinal tuberculosis (TB) is a rare skeletal system localisation of TB in haemodialysis patients. In this paper, a case of Pott's disease with a psoas muscle abscess is reported. The patient had been on the dialysis programme for five years and was complaining of back pain, weight loss and weakness, which were investigated. A thoracolumbar magnetic resonance imaging showed multiple paravertebral abscesses invading the psoas muscle. TB diagnosis was made by microbiological analysis of specimen, which was obtained by fine needle aspiration under computerised tomography guidance.

[SPECIFIC IMMUNE RESPONSE AND PROTECTIVE FACTORS OF NEUTROPHIL GRANULOCYTES IN PULMONARY AND EXTRAPULMONARY TUBERCULOSIS].

An immune response and protective factors of neutrophil granulocytes were comparatively studied in 61 patients with pulmonary tuberculosis, 70 with nephrotuberculosis, and 45 with tuberculous spondylitis. It was shown that there were elevated serum levels of neutrophil cationic proteins in both pulmonary and extrapulmonary tuberculosis. Lower content of intracellular cationic proteins along with suppressed cellular immunity was observed in pulmonary tuberculosis patients with disseminated actively progressive changes. This combination may be regarded as a poor predictor. Higher values of a specific cellular immune response and elevated levels of intracellular cationic proteins of neutrophils were found in extrapulmonary tuberculosis. This was also seen in patients with severe clinical manifestations of the disease. Thus, there is reason to believe that there is a regulatory association of the protective factors of neutrophil granulocytes with cellular immunity in tuberculosis.

[TNF-alpha in prognosis of a recovery of the spinal marrow function in patients with spinal tuberculosis]. / Faktor nekroza opukholi alfa v prognoze vosstanovleniia funktsii spinnogo mozga pri tuberkuleze pozvonochnika.

The TNF-alpha significance in forecasting a degree of recovering of the spinal marrow functions was studied in complicated courses of tuberculous spondylitis in 37 patients with generalized and multiple tuberculosis. The TNF-alpha level in the cerebrospinal fluid was found to be related with a severity of inflammation and of neurological disorders, as well as with a degree of spinal marrow compression and with a speed of regression of postoperative disorders in the spinal marrow. The initial TNF-alpha concentration of > or = 400 pg/ml was indicative of a possibility to ensure a fast regression of postoperative disorders in the spinal marrow, while no complete recovery of spinal-marrow functions was observed in cases the TNF-alpha was < 400 pg/ml.