Cytokines as biomarkers to monitoring the impact of multidrug therapy in immune response of leprosy patients.

Leprosy or Hansen's disease is a chronic infectious disease of the skin and nerves, caused by the intracellular bacilli Mycobacterium leprae. It is characterized by a spectrum of clinical forms depending on the host's immune response to M. leprae. Patients with tuberculoid (TT) leprosy have strong cell-mediated immunity (CMI) with elimination of the bacilli, whereas patients with lepromatous (LL) leprosy exhibit defective CMI to M. leprae. Despite advances in the understanding of the pathogenesis of leprosy and the development of new therapeutic strategies, there is a need for the identification of biomarkers which be used for early diagnosis and to discrimination between different forms of the disease, as prognostic markers. Here, we analyzed the serum levels of IL-1ß, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IFN-γ and TNF in order to address the contribution of these cytokines in late phase of M. leprae infection, and the impact of multidrug therapy (MDT). Our results demonstrated that patients of LL group presented higher expression of serum levels of inflammatory cytokines before MDT, while TT patients presented a balance between inflammatory and regulatory cytokines. MDT changes the profile of serum cytokines in M. leprae infected patients, as evidenced by the cytokine network, especially in TT patients. LL patients displayed a multifaceted cytokine system characterized by strong connecting axes involving inflammatory/regulatory molecules, while TT patients showed low involvement of regulatory cytokines in network overall. Cytokines can be identified as good biomarkers of the impact of MDT on the immune system and the effectiveness of treatment.

Lymph node abscess and cardiac involvement in a patient with nodular lepromatous leprosy (LL) with erythema nodosum leprosum (ENL): a rare occurrence.

With the world's focus on reducing the leprosy patient load to the extent of elimination, finding and reporting the rarer presentations of leprosy becomes important for prompt treatment. Also, these untreated patients may serve as a potential source of infection in community. We report a 35-year old man diagnosed to have lepromatous leprosy and erythema nodosum leprosum with inguinal lymph node abscess and suspected cardiac involvement that proved fatal. We stress the importance of detailed workup to look for associated systemic involvement for timely intervention and favourable outcome.

Prostatic and testicular parameters in lepromatous patients.

OBJECTIVES: To evaluate PSA (Prostate-specific antigen) parameters in patients with lepromatous leprosy (LL). DESIGN: In a retrospective study, 23 male patients with LL were evaluated. PSA parameters (serum total PSA (tPSA), free PSA (fPSA), free-to-total PSA ratio (f/tPSA), PSA Density (PSAD)) were assessed. PSA parameters were compared with a control group. RESULTS: The mean tPSA, fPSA, f/tPSA, prostate volume, and PSAD values of the patient group with LL were 1.87 +/- 0.81 ng/ml, 0.67 +/- 0.29 ng/ml, 0.36 +/- 0.11, 41.08 +/- 23.65 ml and 0.055 +/- 0.037, respectively. The mean tPSA, fPSA, f/tPSA, prostate volume, and PSAD values of the control group were 2.71 +/- 0.91 ng/ml, 0.80 +/- 0.34 ng/ml, 0.30 +/- 0.08, 65.0 +/- 28.73 ml and 0.049 +/- 0.028, respectively. The mean tPSA and prostate volume values were found to be significantly lower in the patient group with LL (p = 0.002 and 0.004, respectively). No significant difference was found between two groups in terms of mean fPSA and PSAD values (p = 0.18 and 0.5, respectively). The mean f/tPSA value was found to be significantly higher in the patient group with LL (p = 0.02). Testes in 16 (69%) patients with LL were bilaterally atrophic. CONCLUSIONS: Serum tPSA values and prostate volumes in the patients with LL were significantly reduced and f/tPSA values were significantly increased. Testicular atrophy in the lepromatous cases might be due to leprosy-related orchitis and associated with a reduction in prostatic volume.

In vitro thalidomide does not interfere with the activation of complement by M. leprae.

Erythema nodosum leprosum (ENL) is an inflammatory reaction that may occur in multibacillary leprosy patients, and thalidomide is the treatment of choice. Its cause and the mechanism by which thalidomide suppresses ENL are not known. In the skin lesions, im- mune complexes and split products of complement are found. The activation of complement could precipitate ENL, and thalidomide could suppress the inflammation by inhibiting the activation of complement. To determine if thalidomide could suppress the activation of complement, we first incubated normal serum with thalidomide and with M. leprae or zymosan. The amount of residual functional complement was then assessed by determining the dilution of serum required to lyses sheep erythrocytes sensitized by rabbit antibodies (CH50 Assay). M. leprae and zymosan activated complement. The residual complement activity in the serum incubated with M. leprae or with zymosan was equivalent to that incubated with M. leprae or zymosan in the presence of thalidomide, hydrolyzed thalidomide and metabolites of thalidomide. Thalidomide did not inhibit the activation of complement by zymosan, a known initiator of complement activation by the alternative pathway, or by M. leprae.

Widespread intradermal accumulation of mononuclear leukocytes in lepromatous leprosy patients treated systemically with recombinant interferon gamma.

Intradermal administration of recombinant interferon gamma (rIFN-gamma) to lepromatous leprosy patients has converted the local histology toward a tuberculoid pattern. However, such changes have been confined to the site of injection. In contrast, in the present study, marked, intradermal accumulation of CD3+, CD4+, CD8+, and CD1a+ T cells and Leu-M5+ mononuclear phagocytes was induced at a distance from the sites of administration, in a dose-dependent manner, by 10 daily intramuscular injections of 10-30 micrograms rIFN-gamma/m2. Mononuclear cell infiltration began within 3 d of onset of rIFN-gamma therapy and persisted at least 8 wk. Intramuscular administration of rIFN-gamma to lepromatous patients receiving concurrent chemotherapy can safely induce widespread histologic features of an upgrading reaction.

Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing.

Recombinant granulocyte/macrophage-colony-stimulating factor (rGM-CSF), prepared from Chinese hamster ovary (CHO) cells and Escherichia coli, was administered to 35 patients with the borderline and polar lepromatous forms of leprosy by the intradermal and subcutaneous routes at doses of 7.5-45.0 micrograms/d for 10 d. With each of these doses and routes, increases in the number of circulating eosinophils were noted. After the intradermal injection, the local skin sites demonstrated zones of roughening and micronodularity that appeared within 24-48 h and persisted for more than 6 d. Reinjection of sites led to enhanced areas of epidermal reaction. GM-CSF prepared from CHO cells was a more potent inducer of this effect. GM-CSF given by the subcutaneous route, at higher doses, failed to initiate these changes. At the microscopic level, the epidermis became thickened (+75%) with increased numbers and layers of enlarged keratinocytes. These contained increased numbers of ribosomes and prominent nucleoli, and were imbedded in a looser meshwork of the zona Pellucida. The modified keratinocytes remained MHC class II antigen negative throughout the course of the response. A major change in the dermis was the progressive accumulation of CD1+, Birbeck granule-positive cells. These Langerhans were recognizable at 48 h after intradermal injection and reached maximum numbers by 4 d. During this period the number of epidermal Langerhans cells remained relatively constant. No increment in dermal Langerhans cells occurred when GLM-CSF was injected by the subcutaneous route. No appreciable increase in the numbers of T cells and monocytes was noted, and granulocytes and eosinophils were largely present within the dermal microvasculature. 4-mm punch biopsies taken from injected sites and adjacent controls were compared in terms of the rapidity of wound healing. 22 of 26 sites demonstrated more rapid filling and hemostasis, whereas four were equivalent to controls. We conclude that rGM-CSF, when introduced into the skin, leads to enhanced keratinocyte growth, the selective recruitment of Langerhans cells into the dermis, and enhanced wound healing of the prepared site. There was no evidence of an enhanced cell-mediated response to Mycobacterium leprae, and bacillary numbers remained unchanged.

A case of lepromatous leprosy with multiple relapses.

We report a case of multiple relapses in a lepromatous leprosy patient after treatment with World Health Organisation (WHO) recommended multibacillary multidrug therapy (MBMDT). The patient responded well to reintroduction of MDT after each relapse.

Erythema nodosum leprosum in Nepal: a retrospective study of clinical features and response to treatment with prednisolone or thalidomide.

INTRODUCTION: Erythema nodosum leprosum (ENL) is an inflammatory reaction, which may occur in the course of leprosy and may result in nerve function impairment and subsequent disability. METHODS: This retrospective study explores demographic and disease specific parameters. Severity of ENL was assessed using the Reaction Severity Scale (RSS). Records of 94 patients were reviewed. The study reports also on the treatment of 76 of these patients who were treated with prednisolone alone or thalidomide in addition to prednisolone. RESULTS Thirty percent of patients presented with ENL at time of diagnosis; 41% developed ENL-reaction in the first year of MDT. Forty-eight percent of patients were treated for ENL-reaction for less than 12 months; 13% for more than 5 years. High RSS-scores correlated with a longer duration of treatment. In group A (prednisolone) 51.7% and in group B (prednisolone and thalidomide) 76.6% of patients were male. Age, leprosy classification, delay of multidrug treatment (MDT) and interval between MDT and first ENL-symptoms did not differ significantly in both groups. Median duration of ENL-treatment was 15 months in group A versus 38 months in group B (P < 0.001). At the start of treatment, ENL-reaction was less severe in group A (RSS = 12) than in group B (RSS = 18; P = 0.003). DISCUSSION: ENL-symptoms may be of help in the early diagnosis and adequate treatment of ENL. Characterisation of (sub) groups of patients with ENL based on presence and severity of symptoms is important for future prospective studies to better evaluate the efficacy of interventions.

Use of fine needle aspiration cytology in leprotic lesions: a report of 4 cases.

BACKGROUND: Although a few studies have shown fine needle aspiration cytology (FNAC) to be a sensitive diagnostic tool in the detection of nerve involvement, its role as an initial diagnostic procedure in pure neuritic leprosy (PNL) and in the detection of skeletal lesions with unusual findings has not been documented before. CASES: Three patients who presented with thickened nerves and a fourth with biopsy-proven lepromatous leprosy with lesions in hand bones underwent FNAC. Of the 3 patients with nerve thickening, 2 had a clinical suspicion or diagnosis of neuritic leprosy, whereas in the third patient a clinical differential diagnosis of a soft tissue tumor or parasitic cyst was considered. FNAC in all 3 cases revealed epithelioid cell granulomas, Langhans giant cells and caseous necrosis. Fites and Ziehl-Neelsen stains were negative for acid-fast bacilli. Cytologic diagnosis of pure neuritic leprosy was made in all 3 cases and confirmed by histopathologic examination. FNAC of skeletal lesions from the fourth patient confirmed involvement of bone with unusual cytologic findings of epithelioid cell granulomas and giant cells along with a significant proportion of foamy macrophages and strong Fites stain positivity. CONCLUSION: FNAC is a simple, useful, minimally traumatic and routinely applicable procedure in the diagnosis of pure neuritic leprosy and leprous osteitis.

Structural and functional changes in the microcirculation of lepromatous leprosy patients - Observation using orthogonal polarization spectral imaging and laser Doppler flowmetry iontophoresis.

Leprosy is a chronic granulomatous infection of skin and peripheral nerves caused by Mycobacterium leprae and is considered the main infectious cause of disability worldwide. Despite the several studies regarding leprosy, little is known about its effects on microvascular structure and function in vivo. Thus, we have aimed to compare skin capillary structure and functional density, cutaneous vasomotion (spontaneous oscillations of arteriolar diameter), which ensures optimal blood flow distribution to skin capillaries) and cutaneous microvascular blood flow and reactivity between ten men with lepromatous leprosy (without any other comorbidity) and ten age- and gender-matched healthy controls. Orthogonal polarization spectral imaging was used to evaluate skin capillary morphology and functional density and laser Doppler flowmetry to evaluate blood flow, vasomotion and spectral analysis of flowmotion (oscillations of blood flow generated by vasomotion) and microvascular reactivity, in response to iontophoresis of acetylcholine and sodium nitroprusside. The contribution of different frequency components of flowmotion (endothelial, neurogenic, myogenic, respiratory and cardiac) was not statistically different between groups. However, endothelial-dependent and -independent vasodilatations elicited by acetylcholine and sodium nitroprusside iontophoresis, respectively, were significantly reduced in lepromatous leprosy patients compared to controls, characterizing the existence of microvascular dysfunction. These patients also presented a significant increase in the number of capillaries with morphological abnormalities and in the diameters of the dermal papilla and capillary bulk when compared to controls. Our results suggest that lepromatous leprosy causes severe microvascular dysfunction and significant alterations in capillary structure. These structural and functional changes are probably induced by exposure of the microvascular bed to chronic inflammation evoked by the Mycobacterium leprae.

Clinico-pathological features of erythema nodosum leprosum: A case-control study at ALERT hospital, Ethiopia.

BACKGROUND: Leprosy reactions are a significant cause of morbidity in leprosy population. Erythema nodosum leprosum (ENL) is an immunological complication affecting approximately 50% of patients with lepromatous leprosy (LL) and 10% of borderline lepromatous (BL) leprosy. ENL is associated with clinical features such as skin lesions, neuritis, arthritis, dactylitis, eye inflammation, osteitis, orchitis, lymphadenitis and nephritis. ENL is treated mainly with corticosteroids and corticosteroids are often required for extended periods of time which may lead to serious adverse effects. High mortality rate and increased morbidity associated with corticosteroid treatment of ENL has been reported. For improved and evidence-based treatment of ENL, documenting the systems affected by ENL is important. We report here the clinical features of ENL in a cohort of patients with acute ENL who were recruited for a clinico-pathological study before and after prednisolone treatment. MATERIALS AND METHODS: A case-control study was performed at ALERT hospital, Ethiopia. Forty-six LL patients with ENL and 31 non-reactional LL matched controls were enrolled to the study and followed for 28 weeks. Clinical features were systematically documented at three visits (before, during and after predinsolone treatment of ENL cases) using a specifically designed form. Skin biopsy samples were obtained from each patient before and after treatment and used for histopathological investigations to supplement the clinical data. RESULTS: Pain was the most common symptom reported (98%) by patients with ENL. Eighty percent of them had reported skin pain and more than 70% had nerve and joint pain at enrolment. About 40% of the patients developed chronic ENL. Most individuals 95.7% had nodular skin lesions. Over half of patients with ENL had old nerve function impairment (NFI) while 13% had new NFI at enrolment. Facial and limb oedema were present in 60% patients. Regarding pathological findings before treatment, dermal neutrophilic infiltration was noted in 58.8% of patients with ENL compared to 14.3% in LL controls. Only 14.7% patients with ENL had evidence of vasculitis at enrolment. CONCLUSION: In our study, painful nodular skin lesions were present in all ENL patients. Only 58% patients had dermal polymorphonuclear cell infiltration showing that not all clinically confirmed ENL cases have neutrophilic infiltration in lesions. Very few patients had histological evidence of vasculitis. Many patients developed chronic ENL and these patients require inpatient corticosteroid treatment for extended periods which challenges the health service facility in resource poor settings, as well as the patient's quality of life.

Blink reflex, H-reflex and nerve-conduction alterations in leprosy patients.

Peripheral nerve lesions are the most important cause of disability in leprosy patients. Electrophysiological studies are used in the diagnosis and prognosis of neuropathy. Nerve conduction is the most frequently used electrophysiological test method to detect neuropathy, although it evaluates only a part of the peripheral nervous system. Blink reflex and H-reflex are electrophysiological tests which evaluate facial and trigeminal nerve function. This study determined the frequencies of blink reflex, H-reflex and motor and sensory nerve conduction alterations in twenty five heterogeneous, clinic patients with lepromatous leprosy and a control group of 20 healthy subjects. Study results showed a decrease in motor and sensory nerve conduction in 40% and 30%, respectively. In blink reflex (BR), right R1 was altered in latency in 20% of patients, left R1 in 20%, right ipsilateral R2 in 16%, left ipsilateral R2 in 20%, and right and left contralateral R2 were altered in 32% of patients. There was an absence of H-reflex in 16% (n = 4) and prolonged latency in 4% (n = 1).

Lepromatous leprosy with bilateral facial nerve palsy and hyperthyroidism.

Bilateral lagophthalmos secondary to facial nerve is extremely uncommon. Further, the aetiology in most of these cases is of central origin unlike the peripheral involvement in leprosy. A patient of lepromatous leprosy (LL) may be euthyroid or hypothyroid on account of leprous involvement of the thyroid gland. A case of LL with bilateral lagophthalmos and hyperthyroidism is reported.

Histopathology of the lepromatous skin biopsy.

The histopathology of lepromatous skin varies according to the cell-mediated immunity of the host against Mycobacterium leprae. In tuberculoid and borderline tuberculoid leprosy, epithelioid noncaseating granulomas predominate, and acid-fast bacilli (AFB) are absent or only rarely present. In borderline lepromatous and lepromatous leprosy, the infiltrate is composed of macrophages with a vacuolar cytoplasm, lymphocytes, and plasma cells. AFB are numerous. Edema inside and outside the epithelioid granulomas, together with the appearance of large giant cells, are the main features of type 1 reactions. A conspicuous neutrophilic infiltrate in the subcutis with or without vasculitis is found in erythema nodosum leprosum. The main histopathologic features of leprosy and its particular forms are discussed in this review.

Clinical aspects of leprosy.

Leprosy is a chronic, infectious disease caused by Mycobacterium leprae. It mainly affects the peripheral nervous system, skin, and certain other tissues such as the reticulo-endothelial system, bones and joints, mucous membranes, eyes, testes, muscles, and adrenals. Leprosy clinical presentation varies from few to widespread lesions. In most patients, early leprosy presents as macular and hypopigmented lesions. This initial clinical presentation is known as indeterminate leprosy and occurs in individuals who have not developed cell-mediated immunity against M. leprae yet. The number of lesions depends on the genetically determined cellular immunity of the patient. Individuals presenting a vigorous cellular immune response and limited humoral immune responses to M. leprae, usually present few skin lesions. Without treatment, those patients tend to evolve into the polar tuberculoid or borderline tuberculoid form of leprosy. Due to the inability to mount an effective cellular-mediated response to M. leprae and the consequent hematogenous spread of the bacilli, some patients may present with numerous and symmetrically distributed hypochromic lesions. Without treatment these patients evolve to a nonresistant form of leprosy, polar lepromatous.

Asymptomatic nerve hypertrophy in lepromatous leprosy: a clinical, electrophysiological and morphological study.

In order to learn more about early nerve lesions observed in leprosy, we performed a clinical, electrophysiological and morphological study in seven patients with untreated lepromatous leprosy, palpably enlarged radial cutaneous nerve and preserved sensation in the corresponding territory. The conduction velocity of the cutaneous radial nerve, which was decreased in all patients, did not significantly differ from that of a group of patients with lepromatous leprosy, hypertrophy of the radial cutaneous nerve and sensory loss. In contrast, the sensory action potential was significantly lower in patients with sensory loss, which demonstrates that axon loss is more important than demyelination in producing sensory loss. In all patients nerve enlargement was due to thickening of the epineurium and of the perineurium subsequent to inflammatory infiltrates and proliferation of fibroblasts and perineurial cells. In several fascicles, the inflammatory infiltrates and the infected cells infiltrated endoneurial connective tissue septa and blood vessels. Mycobacteria leprae were abundant in perineurial cells, fibroblasts, macrophages, Schwann cells and endothelial cells, and lymphocytic vasculitis present in all cases. The average density of myelinated fibres was 2600 SD 880 fibres/mm2 (control: 7700 fibres/mm2), with marked differences between individual fascicles, versus 420 fibres/mm2 in patients with nerve hypertrophy and sensory loss (range 0-2080 fibres/mm2). Single fibre preparations showed that segmental demyelination predominated in two patients, axonal degeneration in one, while inflammatory infiltrates and proliferation of connective tissue adhering to individual fibres were prominent in the others.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of untreated chronic plastic iridocyclitis on intraocular pressure in leprosy patients.

The intraocular pressures of a total of 286 eyes of patients with lepromatous and borderline lepromatous leprosy who never had regular ophthalmological care or local eye treatment were measured. The patients were categorised according to the type of leprosy they had, and the eyes were categorised as without or with chronic plastic iridocyclitis. In patients with lepromatous and borderline lepromatous types of leprosy the intraocular pressure was significantly lower in eyes with chronic plastic iridocylitis 10.1 (3.6) mmHg than in both unaffected eyes 11.0 (3.2) mmHg and control eyes 13.5 (2.5) mmHg. It has been shown that chronic plastic iridocyclitis which remains untreated for years results in a lower intraocular pressure than normal.

Evaluation of chemiluminescence, procoagulant activity and antigen presentation by monocytes from lepromatous leprosy patients with or without reactional episodes.

In this study, we evaluated the activity of peripheral blood mononuclear cells (PBMC), isolated from treated and untreated lepromatous leprosy patients, from lepromatous leprosy patients during and after reactional episodes (erythema nodosum leprosum (ENL) and reversal reaction (RR)), and from normal healthy individuals. We determined reactive oxygen intermediate (ROI) production, procoagulant activity (PCA) and HLA-DR antigen expression of monocytes, besides lymphoproliferation, both in the presence and absence of various stimulatory agents. Phorbol myristate acetate (PMA) stimulated ROI production by monocytes from all the groups studied, with patients during reactional episodes (ENL and RR) showing a significantly higher response (p < 0.009 and p < 0.00001). Irradiated Mycobacterium leprae, although having little effect when added alone, strongly suppressed PMA-stimulated ROI production. Muramyl dipeptide (MDP) had no influence on either basal or on PMA-induced ROI production. Basal monocyte PCA, as well as M. leprae or concanavalin A (ConA)-induced monocyte PCA was comparable in monocytes from all the groups studied. ConA was able to induce mitogenic activity in mononuclear cells isolated from all the groups studied. M. leprae, although stimulatory for normal individuals, did not induce lymphoproliferation in lepromatous leprosy patients, except for cells from patients during RR, which responded equally to M. leprae and to ConA. The absence of M. leprae-induced lymphoproliferation in lepromatous leprosy patients is not caused by the lack of basal HLA-DR expression, as PBMC from all individuals studied showed the same level of this antigen. Our results suggest an increase of spontaneous or PMA-induced monocyte activity, as detected by ROI production, during the reactional episode; addition of M. leprae suppressed this response. The increase in monocyte activity could be correlated with the increase of lymphoproliferation response to M. leprae during RR, but not during ENL. The importance of a possible immune suppressive action of M. leprae is discussed.