Complement Component C1q as Serum Biomarker to Detect Active Tuberculosis.

Background: Tuberculosis (TB) remains a major threat to global health. Currently, diagnosis of active TB is hampered by the lack of specific biomarkers that discriminate active TB disease from other (lung) diseases or latent TB infection (LTBI). Integrated human gene expression results have shown that genes encoding complement components, in particular different C1q chains, were expressed at higher levels in active TB compared to LTBI. Methods: C1q protein levels were determined using ELISA in sera from patients, from geographically distinct populations, with active TB, LTBI as well as disease controls. Results: Serum levels of C1q were increased in active TB compared to LTBI in four independent cohorts with an AUC of 0.77 [0.70; 0.83]. After 6 months of TB treatment, levels of C1q were similar to those of endemic controls, indicating an association with disease rather than individual genetic predisposition. Importantly, C1q levels in sera of TB patients were significantly higher as compared to patients with sarcoidosis or pneumonia, clinically important differential diagnoses. Moreover, exposure to other mycobacteria, such as Mycobacterium leprae (leprosy patients) or BCG (vaccinees) did not result in elevated levels of serum C1q. In agreement with the human data, in non-human primates challenged with Mycobacterium tuberculosis, increased serum C1q levels were detected in animals that developed progressive disease, not in those that controlled the infection. Conclusions: In summary, C1q levels are elevated in patients with active TB compared to LTBI in four independent cohorts. Furthermore, C1q levels from patients with TB were also elevated compared to patients with sarcoidosis, leprosy and pneumonia. Additionally, also in NHP we observed increased C1q levels in animals with active progressive TB, both in serum and in broncho-alveolar lavage. Therefore, we propose that the addition of C1q to current biomarker panels may provide added value in the diagnosis of active TB.

Mapping genetic variants in the CFH gene for association with leprosy in Han Chinese.

Complement factor H (CFH) is an essential regulator in the homeostasis of the complement system that plays multiple roles in leprosy. We previously reported a preliminary association of CFH with leprosy, but potentially causal variants remain to be identified. In this study, we performed a fine-mapping association analysis in 1110 individuals (527 leprosy patients and 583 controls) followed by bioinformatic analyses. We identified no association of typical missense CFH variants with leprosy and factor H-binding protein was not detected in Mycobacterium leprae. However, robust associations (PBonferroni<0.003) of several CFH intronic tag single-nucleotide polymorphisms with leprosy were observed. Expression quantitative trait locus analysis showed that these leprosy-protective alleles were associated with higher CFH level and lower CFHR3 (complement factor H-related 3) level. Our results indicated that CFH variants may contribute to leprosy pathogenesis through altering CFH expression, leading to regulation of complement activity rather than mediating immune evasion by bacteria binding.

Serum proteome of leprosy patients undergoing erythema nodosum leprosum reaction: regulation of expression of the isoforms of haptoglobin.

Validated proteome profile allows better understanding of disease progression, subtype classification, susceptibility patterns, and disease prognosis. Leprosy is a spectral disease, with clinically, histologically, immunologically, and bacteriologically distinguishable subtypes. In addition, a significant fraction of patients undergo immune mediated reactions even after multidrug therapy (MDT). Erythema nodosum leprosum (ENL) is an immune complex mediated reactional condition in leprosy, characterized by a systemic inflammatory condition afflicting borderline lepromatous (BL) and lepromatous leprosy patients (LL). In this study, we have analyzed serum proteome of leprosy patients undergoing ENL reactions and compared it with that of healthy noncontact controls. Depletion of albumin and immunoglobulin G (IgG) was optimized using Aurum serum protein mini kit (Bio-Rad), and then two-dimensional gel electrophoresis (2-DE) of these serum samples was performed. Differentially expressed proteins were identified by MALDI-TOF and MALDI-TOF MS/MS mass spectrometry. Significant increase in one of the isoforms of alpha2 chain of haptoglobin was observed in ENL condition. In addition, haptoglobin phenotype was determined for healthy controls and leprosy patients. Hp 0-0 phenotype was detected in 21.4% of the ENL patients undergoing treatment, which on follow up examination showed typable phenotype, thus showing a condition of acquired anhaptoglobinemia. Since ENL still remains a threat to leprosy disease management, the above findings may provide new insights in understanding the development and progression of this inflammatory condition.

Cutaneous vasculitis as a presenting feature of multiple myeloma: a report of 2 cases.

We report two male patients who presented with symmetrical, painful purpura that evolved into bullae and necrotic ulcers, predominantly on the extremities, over two months in spite of conventional therapy including oral steroids. Examination showed livedoid and purpuric patches with necrotic centers in starburst pattern over the extremities and buttocks. The first case also had similar lesions over the ears. The clinical presentation and the histopathological examination suggested a diagnosis of necrotizing leukocytoclastic vasculitis (LCV). Blood testing ruled out connective tissue disease, hepatitis B or C infection or streptococcal infection as underlying cause of vasculitis. Serum antinuclear factor, antineutrophilic cytoplasmic antibody and anticardiolipin anticoagulant were negative in both cases. Cryoglobulins were positive in case 2. An incidental finding was raised serum proteins and globulins in case 2. Further investigations revealed M band on electrophoresis and features of multiple myeloma on bone marrow biopsy in both cases. These cases emphasize the importance of simple investigations like serum proteins in the evaluation of LCV.

Protein profile in leprosy.

Serum proteins and plasma fibrinogen were estimated in 103 patients in various groups of leprosy and 52 patients of reactional leprosy. Total proteins, serum globulin and fibrinogen showed significant rise while serum albumin showed fall over the immunological spectrum from TT to LL. Type II reactional leprosy similarly revealed significant rise in globulin and fibrinogen. The comparison of these parameters between most of the comparable groups of leprosy was statistically significant. ENL patients after complete subsidence of reaction and after steroid treatment showed significant decrease in these protein fractions, thus conferring some prognostic implication on these tests.

Serum zinc, copper, magnesium, proteins and superoxide dismutase in leprosy patients on multidrug therapy--a follow-up study.

Serum zinc, copper, magnesium, total proteins and albumin-globulin fractions and superoxide dismutase (SOD) were estimated in 80 untreated patients with TT/BT/BL/LL type of leprosy and in 40 controls. The investigations were repeated on day 30, 60 and 120 after starting multidrug therapy (MDT-WHO) on the patients. Serum zinc was significantly lowered in all types of leprosy on days 0 and 30. Serum copper was significantly raised in all types of leprosy. This was not significant in BT/TT cases on 60, 120 days. There was a correlation between serum zinc and copper levels and the severity and type of leprosy. The lowering of serum magnesium values were not significant. With therapy, there was a shift of all the three elements towards normal values. Serum total proteins reduction was not significant. Serum albumin was significantly lowered in all types of leprosy. Serum globulin was significantly raised in all types of leprosy. This rise in TT/BT was not significant on day 60 and 120 after starting treatment. Serum SOD was significantly reduced in all the untreated cases. It gradually increased with the clinical improvement under MDT.

Altered calcium-binding ability of plasma proteins as the cause of hypocalcemia in lepromatous leprosy.

This paper reports a study performed on 10 lepromatous leprosy outpatients and on the same number of age- and sex-matched contacts. All of the lepromatous patients were hypocalcemic, but plasma levels of ionized calcium and the acid-base status were normal. The average daily food intake assessed through a questionnaire revealed adequate nutrition of patients and controls. Plasma proteins and 1,25-dihydroxyvitamin D3 and intestinal absorption of calcium were discarded as the causes of the hypocalcemia. In vitro experiments designed to investigate the effect of hydrogen ion concentration on the equilibrium between calcium ion and proteins revealed that, at normal pH values, plasma proteins from lepromatous leprosy patients bind a smaller fraction of total plasma calcium than those from controls. This phenomenon produces a normal concentration of ionized calcium that determines a normal parathyroid status as indicated by the normal urinary excretion of hydroxyproline and plasma concentrations of alkaline phosphatase (total and bone isoenzyme) and tartrate-resistant acid phosphatase.

Activation of complement by circulating immune complexes isolated from leprosy patients.

Circulating immune complexes isolated from different types of leprosy sera as polyethylene glycol (PEG) precipitates were found to be efficient activators of the alternative pathway of complement. PEG precipitates from BL/LL leprosy patients and those with erythema nodosum leprosum were found to activate both the classical pathway and the alternative pathway of complement efficiently, while PEG precipitates from TT/BT leprosy patients and borderline tuberculoid patients in reaction were found to active the alternative pathway of complement but not the classical pathway. No significant differences were observed between the PEG precipitates from reactional and nonreactional TT/BT and BL/LL patients in their complement activating ability.

Clinical significance of changes in serum proteins, immunoglobulins, and autoantibodies in leprosy.

Changes in the level of acute phase reactants such as C-reactive protein (CRP), serum globulins, and autoantibodies have been reported previously in patients with leprosy, particularly at the lepromatous end of the spectrum. The clinical significance of these findings was investigated by comparing the same parameters of humoral immune function in populations of Australian Aboriginals with stable treated leprosy and relevant contact groups including a) noninfected European sporadic contacts and b) healthy Aboriginal relatives of patients with confirmed leprosy. Raised levels of CRP and immunoglobulins and the higher frequency of autoantibodies seen in leprosy patients compared with sporadic contacts are probably related to differences in the incidence of nonleprous infection rather than to leprosy per se. Comparable results were obtained in the leprosy patients and their family contacts. The data highlight the need to use antigen-specific assays for determining the significance of changes in acute phase reactants and for distinguishing between the primary and secondary effects of Mycobacterium leprae infection.

Studies on serum proteins in leprosy by polyacrylamide gel electrophoresis (page)--I.

Serum protein pattern was studied in the leprosy spectrum, their contacts and in normal individuals by employing polyacrylamide gel electrophoresis. Sera from 80% of untreated BL/LL, 70% of untreated TT/BT patients and 67% of contacts have shown dysproteinaemia either for 232 kD or for 175 kD or for both these proteins together. Tendency of these proteins to return to normal levels was observed after treatment. But both these proteins come back to normal levels only after subsidence of the disease.

Study of rheumatic manifestations and serologic abnormalities in patients with lepromatous leprosy.

We investigated the rheumatic and laboratory features associated with rheumatic syndromes in 32 patients with lepromatous leprosy. Twenty-seven (84%) developed a broad range of rheumatic manifestations, the most common being the presence of arthritis which was symmetric and polyarticular, resembling rheumatoid arthritis. The laboratory abnormalities included an elevated sedimentation rate in 32 cases (100%), a positive rheumatoid factor in 6 (18.7%), and antinuclear antibodies in one (3.1%). A careful history and the recognition of rheumatic manifestations will help in the identification of this type of leprosy.

Leprosy in a mangabey monkey--naturally acquired infection.

Naturally acquired leprosy was detected in an otherwise normal "sooty" mangabey monkey (Cercocebus atys). This animal was imported from West Africa in 1975 and developed clinical symptoms of leprosy in 1979. Histopathologic findings were those of subpolar-lepromatous to borderline-lepromatous leprosy in the Ridley-Jopling classification. The disease was progressive, with crippling neuropathic deformities of the hands and feet. The disease regressed under specific therapy. The etiologic agent was identified as Mycobacterium leprae by the following criteria: invasion of nerves of host, staining properties, electron microscopic findings, noncultivable on mycobacteriologic media, DOPA-oxidase positive, lepromin reactivity, infection patterns in mice and armadillos, sensitivity to sulfone, and DNA homology. We believe the animal acquired the disease from a patient with active leprosy. The mangabey monkey offers promise as a primate model for leprosy, and adds a third reported species to animals with naturally acquired leprosy.

Experimental murine leprosy: a biochemical study emphasizing lysosomal enzyme changes in vivo and enzyme secretion by macrophages in vitro.

This study was undertaken to identify biochemical alterations in serum, lymphoid organs, and peritoneal macrophages (PM) which reflect the histopathology of experimental Mycobacterium lepraemurium (MLM) infection in mice. A significant increase of acid phosphatase, beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, and lysozyme was found in serum, spleen, and liver homogenates of mice infected intraperitoneally (ip) with MLM. PM from infected mice showed a substantially greater rate of secretion of beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, and acid phosphatase than PM from normal mice. There was, however, no significant difference in the ability of PM from BALB/c and C57BL/6 mice to secrete such enzymes in vitro. There was also a significant increase in all these enzymes in PM in the early stage of infection but they dropped to values lower than normal in the advanced stage of infection despite the fact that such cells increased in size and protein content as the infection progressed. Infected mice were also found to have progressively elevated levels of serum lactic dehydrogenase, glutamic oxaloacetic, and glutamic pyruvic transaminases which indicated damages of hepatocytes and other tissues. Values of other blood components were also reported. Both BALB/c and C57BL/6 strain of mice, which are susceptible to the ip route of MLM infection, showed an indistinguishable pattern of biochemical alterations as reflected by their similar histopathological changes in various organs. BALB/c mice, which are still susceptible to subcutaneous (sc) route of infection showed similar characteristic changes in various serum components as before. In contrast, C57BL/6 mice, which are resistant to MLM infection sc, showed insignificant alterations in most of these biochemical parameters.

Monocyte-derived soluble suppressor factor(s) in patients with lepromatous leprosy.

Peripheral blood monocytes from polar lepromatous leprosy (LL) patients were unable to support Mycobacterium leprae-induced in vitro lymphoproliferation of HLA-D-matched T cells from tuberculoid leprosy subjects, whereas those from responder individuals were able to do so. Monocyte-rich adherent cells from untreated LL patients released de novo soluble factors which inhibited antigen-induced lymphoproliferation to a greater extent and mitogenic responses to a lesser extent. Suppressive activity varied in different LL patients. However, the degree of suppression was similar in soluble factors obtained de novo and after treatment of adherent cells with heat-killed and freshly extracted, cryopreserved M. leprae. Treated patients showed less inhibition with de novo released soluble factors (27 +/- 7.7%) as compared to parallel soluble factors obtained after antigen treatment (44 +/- 4.8%) or with de novo soluble factors from untreated LL patients (62 +/- 14.2%). Similar supernatants from tuberculoid individuals showed no or insignificant effects on antigen-induced lymphoproliferation. The suppressive activity of LL soluble factors was produced for up to 72 h, was heat stable at 56 degrees C for 30 min, was indomethacin resistant, and resided in the greater than 25,000 molecular weight fraction.

The effect of clofazimine on liver function tests in lepra reaction (ENL).

Twenty patients with suspected DDS resistance and repeated attacks of lepra reactions were selected for the study. Clofazimine was administered in different doses over a period of 12 months. Elevated levels of transaminases and Alkaline phosphatase prior therapy attained values to near normalcy. Progressive fall in serum Bilirubin and Proteins with normal A/G ratio at the end of therapy was also observed. Clofazimine by its anti-inflammatory and antibacterial action could inhibit the process of liver damage and happened to have minimal deleterious effect on liver by studying the liver function tests.

Identification of components of IC purified from human sera. II. Demonstration of mycobacterial antigens in immune complexes isolated from sera of lepromatous patients.

Immune complexes have been purified from sera of patients with lepromatous leprosy, using solid phase conglutinin and analysed by SDS-PAGE. Some of their components have been immunologically identified after electrophoretic blotting on nitrocellulose. First, immunoglobulins, complement components (C1q, C1s, C3) and CRP were found in IC. Secondly, one mycobacterial antigen of 67 kD was directly identified in IC while two other components (20 kD and 14.4 kD) of possible M. leprae origin were also found in IC. This study suggests that lepromatous patients develop a good antibody response against some M. leprae antigens (33 kD and 12 kD), which are rapidly eliminated from circulation, while other M. leprae antigens (e.g. 67 kD) can persist in relative antigen excess, within circulating IC.