Immunotherapy with Mycobacterium vaccae and peripheral blood flow in long-treated leprosy patients, a randomised, placebo-controlled trial.

OBJECTIVE: to evaluate immunotherapy as a means of improving peripheral blood flow in chronic leprosy patients. DESIGN: this was a double-blind, randomised, placebo-controlled, clinical trial. MATERIALS: heat-killed Mycobacterium vaccae 1mg plus 0.02 microg Tuberculin protein per 0.1 ml dose in borate buffer, with saline as placebo. Those studied were 92 long-treated residents of a leprosy centre in Iran, 10 of their healthy children and 10 staff members. Evaluation employed the Perimed PF2, Laser-Doppler Flowmeter, a platinum skin thermistor, and a thermal sensibility tester. METHODS: single intradermal injections of test or placebo were given to 103 patients 18 months before the blinded evaluation. Fingerpulp blood flux was measured in controlled conditions and vasomotor reflexes and skin sensation to touch, pain and heat were evaluated in 45 and 47 patients in the placebo and M. vaccae groups, respectively, and in 20 healthy control persons. RESULTS: Laser-Doppler flux, skin temperature, vasomotor reflexes and sensation were impaired in leprosy patients. Immunotherapy improved (p < 0.05) Laser-Doppler flux, skin temperature and temperature sensation. CONCLUSIONS: immunotherapy, given 18 months earlier, significantly improved blood flow and temperature sensation, in fully-treated, chronic, leprosy patients. The same principles might be employed in other conditions of reduced peripheral blood flow.

Sympathetic vasomotor dysfunction in leprosy patients: comparison with electrophysiological measurement and qualitative sensation testing.

Testing of skin vasomotor reflexes (VRs) by laser Doppler flowmetry (LDF) is now a recognised method of measuring peripheral dysautonomia. To assess its specificity as an indicator of impairment to unmyelinated autonomic fibres, VR testing at the fingerpulp was compared with standard qualitative sensation (QST) and with sensory electrophysiological (SNVC) measurements in 39 Iranian leprosy patients. There was a significant relationship between VR and SNCV values (but not QST): these were jointly measurable in 38.5% of fingers, and jointly absent in 35.3% of fingers which also showed significantly reduced LDF perfusion and skin temperatures. However, in 10.3% of fingers, predominantly index and otherwise apparently healthy, VRs were absent but SNCV present, suggesting early sub-clinical autonomic impairment. In a further 16% of fingers, predominantly ulnar and with poor microcirculation, intact (though impaired) VRs could be recorded despite the absence of SNCV responses, suggesting sparing or regeneration of these fibres. This evidence suggests that where there is heterogeneity of nerve damage a combination of VR and electrophysiological testing can indicate the functional status of distinct fibre types.

Perfusión Doppler con laser ionfrarrojo y cercano al rojo en manos de pacientes de lepra en España

Se ha empleado una nueva técnica de perfusión Doppler con láser a las longitudes de onda, rojo (663 nm) y cercano al rojo (780 nm) para obtener imágenes del flçujo sanguíneo de los dedos de pacientes de lepra y vonluntarios sanos en el Sanatorio de Fontilles. La técnica permite medir el flujo en las estructuras dérmicas y subdérmicas y podría ser útil para localizar deterioro microcirculatorio. Se pueden medir niveles muy elevados de riego en la pulpa de los dedos a la temperatura ambiente de Fontilles (23-6.ºC), ya debido a una abundante anastomosis arteriovenosa, desempeñan un papel termorregulador. Sin embargo en muchos pacientes, incluyendo los que no presentan un compromiso clínico en las manos, el riego sanguíneo y temperaturas cutáneas estaban significativamente reducidas, confirmando hallazgos previos en pacientes de lepra en India e Iran. Los hallazgos preliminares indican que estas disminuciones de riego no están localizadas a una región anatómica pero pueden afectar todos los dedos evaluados de un individuo sugiriendo un deterioro más generalizado de la función termoregulatoria. Mientras que esto puede implicar un mecanismo central, también se podría explicar por una vasculitis sub-cutánea continua de los dedos. Nuestros trabajos anteriores indicando una asociación entre pérdida de sensación y reducción del flujo sanguíneo, también sugieren factopres neurales como una depleción de neuropéptidos sensorilaes.

Effects of treatment on the histopathology of leprosy.

AIMS: To identify the histological changes in leprosy skin lesions over the first few weeks after the start of leprosy treatment and to examine their relationship to reversal reaction. METHODS: Sequential skin biopsy during treatment with multiple drug therapy. In this study, a series of 28 patients was studied, from whom two or more biopsies were taken at two week intervals. Fourteen patients had paucibacillary leprosy (PBL) and 14 had multibacillary leprosy (MBL). RESULTS: In most cases, granuloma fraction and bacterial index fell during treatment, the bacterial index being less sensitive than the granuloma fraction. Since the biopsies were fixed in buffered formalin and processed through to paraffin wax, little immunohistochemistry was feasible. However, there was strong evidence of immune activation, with increased expression of HLA-DR in the granulomas of MBL and PBL cases: the epidermis also expressed HLA-DR in several patients. Such changes may reflect gamma IFN production from granuloma lymphocytes. Patients with reversal reaction often showed HLA-DR expression on admission which decreased with corticosteroid treatment. CONCLUSIONS: The results suggest that activation of cell mediated immunity in leprosy lesions occurs during treatment with multiple drug therapy and may not be restricted to those with clinical evidence of reversal reaction.

Circulation and sensation at the fingertips of claw hands.

Measurements of skin blood flow (by laser Doppler flowmetry) and temperature were made under environmental conditions promoting peripheral vasodilatation at the fingertips of a disfigured 'clawed' hand in 12 leprosy patients long-resident at Baba Baghi Leprosy Hospital, Tabriz, Iran. Sensory function was assessed by measuring the responses to light touch, pain and temperature of each finger, and peripheral autonomic function was gauged by estimating palmer sweating and by measuring skin vasomotor reflexes in response to inspiratory gasp. In 2 patients all measured fingers had laser Dopper flux (LDFlux) values and skin temperatures lower than the 95% confidence limits for the mean of 20 healthy controls, i.e. were impaired; in 2 patients all fingers had normal values for LDFlux and temperature; and in 8 patients there was a combination of impairment with most fingers normal for these parameters but with the small finger most commonly impaired. There were 10 (67%) fingers with impaired LDFlux and temperature values who had significant sensory impairment, whereas only 5 (18%) of the fingers with normal LDFlux values and temperatures had a similar sensory deficit. Overall, the fingers with the most impaired sensation had significantly (P < 0.05) lower LDFlux and temperature values than those with no sensory deficit. Microcirculatory impairment was not related to disordered skin vasometer reflexes or dysfunction of sweating. We concluded that the relationship between motor (skeletal muscle) nerve paralysis and any subsequent sensory neuropathy and/or microcirculatory impairment is more complex than might be expected from previous understanding of the disease.

Heat-stable opsonins in tuberculosis and leprosy.

We have examined heat-stable opsonins to 4 species of gamma-irradiated mycobacteria (M. tuberculosis (H37Rv), M. avium (28A), M. scrofulaceum and M. leprae (cd 103)) in complement-depleted sera collected from Indonesian subjects with tuberculosis (106 patients),-leprosy (24 patients) and controls (40 hospital workers and 41 factory workers) indirectly by microtitre plate chemiluminescence (CL) assay and compared the results with antibody levels. The results indicate that there is a wide range of opsonic capacity for mycobacteria in complement-depleted sera. There was a poor correlation between the opsonic capacity as measured by CL and the anti-mycobacterial antibody content of sera measured by ELISA, suggesting that anti-mycobacterial antibody has little influence on the uptake of mycobacteria. However, a non-specific heat-stable opsonin appears to be present in some sera. Conversely, some sera from tuberculosis or leprosy patients suppress the production of reactive oxygen species from normal phagocytes in vitro when stimulated with M. tuberculosis. The relevance of this inhibition and the presence of heat-stable opsonins to the pathogenesis of tuberculosis have yet to be determined, but it is possible that the presence of opsonins may inhibit dissemination of tubercle bacilli to other organs.

Vasomotor reflexes in the fingertip skin of patients with type 1 diabetes mellitus and leprosy.

Fingertip skin blood flow was measured by laser Doppler flowmetry (as LDflux) under environmental conditions promoting vasodilation in Scottish patients with diabetes mellitus and Indian patients with leprosy. The reflex control of fingertip blood flow was assessed by measuring the reduction in LDflux induced by deep inspiratory gasp (IG) and cold challenge (CC) of immersing the contralateral hand in cold water. The uncomplicated diabetic patients showed normal vasomotor reflexes and an increased, though non significant, LDflux level (p < 0.06). The patients with diabetic neuropathy had resting LDflux levels significantly less than the uncomplicated group and also had substantial impairment of both IG and CC reflexes. Those with retinopathy (but no clinically apparent neuropathy) had LDflux within the normal range, but they showed minor evidence of impairment of the vasomotor reflexes. The uncomplicated newly registered leprosy patients had reduced LDflux and substantial impairment of CC reflexes. These changes were more marked in newly registered leprosy patients with clinical evidence of neuropathy. Leprosy patients with long-standing neuropathy requiring orthopaedic treatment had LDfluxes so greatly reduced that measurement of vasomotor reflexes was not practicable. The CC reflex was more severely affected than the IG reflex and more frequently absent in leprosy patients, possibly because of associated sensory neuropathy affecting the afferent limb of this response. Thus laser Doppler flowmetry can detect impairment of reflex control of fingertip blood flow in both diabetes mellitus and leprosy, but there are functional differences in the pattern of autonomic impairment between the diseases, suggesting differences in the pathogenesis of nerve damage.

Cold fingers in leprosy.

Under conditions of maximal thermoregulatory peripheral dilatation, most healthy subjects (both Indian and European) showed raised blood flow in the fingertips (measured by laser Doppler flowmetry) where the skin temperature is only slightly lower than the core body temperature. Most borderline lepromatous (BL) leprosy patients had much colder fingers and the blood flow was slow: borderline tuberculoid (BT) patients had skin temperatures similar to those seen in healthy subjects, but their fingertip blood flow was reduced relative to that in control subjects. The occurrence of cold fingers and slow blood flow was clearly associated with evidence of sensory impairment to light touch, pressure and temperature. Slower fingertip blood flow was strongly associated with impairment of vasomotor control in this anatomical region, suggesting that both may be a consequence of leprosy peripheral neuropathy, at least in patients with early leprosy, but it is likely that leprosy arteriopathy may contribute to the lowered peripheral perfusion in advanced cases. It is suggested that the simple clinical sign of cold fingers may be of value in the preliminary assessment of patients presenting at any leprosy control clinic in the tropics.

Impairment of fingertip vasomotor reflexes in leprosy patients and apparently healthy contacts.

Fingertip blood-flow velocity and its control by vasomotor reflexes were studied in leprosy patients and in healthy controls with a laser Doppler flowmeter. In newly registered patients, the flow was significantly lower than in the healthy controls, and even lower values were recorded in the long-standing patients with lower limb ulcers and/or deformity. The newly registered patients showed substantially impaired vasomotor reflex responses in the fingertips to cold challenge of the opposite hand or deep inspiratory gasp. Low blood flow and impairment of vasomotor reflexes were more prominent in those leprosy patients who showed clinical evidence of neuropathy and/or histological evidence of reaction in a punch biopsy of leprosy skin lesions. This aspect of dysautonomia to cold challenge was particularly prominent in apparently healthy, fully treated ex-patients. There was an unexpectedly high prevalence of impairment of vasomotor reflexes in newly registered and apparently healthy, adequately treated leprosy patients. The method is very sensitive, and it remains to be established whether the lesions it detects are nonprogressive residues, or previous nerve damage, or an indication of on-going nerve damage. A minority of leprosy contacts showed impairment of vasomotor reflexes. Those with two or more affected fingers were more likely to have had a higher level of exposure to Mycobacterium leprae than those with one or no affected fingers. The cause of this unexpected impairment of fingertip vasomotor reflexes in a minority of leprosy control workers has not yet been determined.

Antibodies to Mycobacterium tuberculosis-specific epitopes in lepromatous leprosy.

Sera from patients with leprosy or tuberculosis and healthy subjects have been analysed for the presence of antibodies to four species-specific mycobacterial epitopes, four different viruses and five autoantigens. Antibodies to the Mycobacterium leprae-specific 35-kD protein and phenolic glycolipid I epitopes were not present in patients with active pulmonary tuberculosis. In contrast, antibody levels to species-specific epitopes of the 38-kD and 14-kD antigens M. tuberculosis were significantly elevated in patients with lepromatous leprosy. Neither of the two antigens is cross-reactive with M. leprae at the B cell level. However, it was considered that cross-reactive helper T cells could recall the response of M. tuberculosis-specific memory B cells, which had been primed through prior self-healing tuberculous infection. As an alternative explanation, the possible role of polyclonal B cell stimulation was considered. This seemed unlikely, however, since: (i) antibody levels to autoantigens, except anti-smooth muscle, were not elevated, and (ii) antibody levels to four distinct viruses, unlike those to all mycobacterial epitopes, showed no correlation with titres, to M. tuberculosis-specific epitopes.

Impairment of vasomotor reflexes in the fingertips of leprosy patients.

A method is described for eliciting fingertip vasomotor reflexes by inspiratory gasp and contralateral hand cold challenge. The results of the two tests are reproducible on replicate testing and, when taken together, have proved reliable for detection of impairment of autonomic reflexes in 10 newly registered leprosy patients who did not have any obvious deformity. Similar, but less severe, impairment of vasomotor reflexes was noted in a group of 10 fully treated, apparently cured ex-leprosy patients, none of whom showed clinically obvious neuropathy. Both the new patients and the ex-patients were significantly different from healthy contacts and from healthy Europeans, who were indistinguishable by this test. Evidence is presented suggesting that impairment of these vasomotor reflexes is mainly due to damage to the efferent pathway in the peripheral nerves. The method might prove valuable for investigation of early nerve damage in new patients or during reversal reactions in leprosy at a stage before irreversible damage is done.

A quantitative study of the relationship between systemic and histological parameters of immunity in individual leprosy patients.

A group of 52 untreated leprosy patients were examined to determine the relationship between local and systemic immunological parameters across the clinico-pathological spectrum. The Ridley-Jopling classification, bacterial index (BI), and granuloma fraction (GF) were assessed in biopsies from 40 cases. The densities of apoptoses, mitoses, and plasma cells were also measured. Systemic immunity to mycobacteria was assessed by skin tests with leprosin A and PPD, and by measurement of the serum antibody responses to Mycobacterium leprae, M. tuberculosis, and M. scrofulaceum. The serum responses to phenolic glycolipid-I (PGL-I) of M. leprae was assessed using a glycoconjugate which mimics an immunodominant epitope. The serum antibody levels and skin test results showed the expected inverse relationship. The BI within lesions showed an inverse correlation with the skin test results, but none of the other histological parameters studied showed a significant relationship with the other measurements of systemic immunity. Our findings suggest that the inverse relationship between delayed-type hypersensitivity and humoral immunity in leprosy patients, which is strong in groups of patients across the leprosy spectrum, is less strong in individual patients than is often thought. The lack of correlation of many histological and systemic parameters suggests that local factors modulate systemic immunity in the pathogenesis of leprosy lesions.

Total and anti-mycobacterial IgE levels in serum from patients with tuberculosis and leprosy.

A radioallergosorbent assay (RAST) was developed and used to determine the levels of IgE antibodies to soluble antigens of Mycobacterium tuberculosis (BCG vaccine strain) in sera from patients with tuberculosis and leprosy and in healthy control subjects. Total IgE levels in the same sera were quantitated with a commercial radioimmunoassay kit. Patients with tuberculosis and leprosy had higher total and specific IgE levels than the control groups but the overlap of levels in patients and controls was too great to render the difference diagnostically useful. Specific IgE levels were elevated in both tuberculosis and leprosy patients, suggesting that this antibody response is towards the shared mycobacterial antigens. No differences in total or specific IgE levels were found between healthy hospital workers occupationally exposed to patients with tuberculosis and factory workers who are not exposed.

Suppress of the increase in free cytosolic calcium during the inhibition of T-cell activation by an autoantibody present in the serum of leprosy patients.

A serum factor, believed to be an IgG autoantibody, in certain patients with lepromatous leprosy inhibits the proliferation of mitogen-stimulated lymphocytes. To investigate which stage of the cell cycle was inhibited, we examined the effect of these sera on the kinetics of lymphocyte activation induced by several mitogenic agents: phytohaemagglutinin (PHA), the calcium ionophore A23187, the phorbol ester phorbol myristate acetate (PMA) and purified protein derivative of BCG (PPD). Seven out of 54 sera tested were found to inhibit PHA-stimulated proliferation. Inhibitory sera and to a lesser extent serum IgG from leprosy patients were capable of suppressing the increase in free cytosolic calcium normally observed immediately after PHA stimulation. Subsequent stages of the cell cycle, increase in cell size, the expression of the IL-2 receptor and increase in DNA were also suppressed. The inhibitory sera was not toxic and, if addition of the sera was delayed, would not inhibit lymphocytes that had already entered the cell cycle. Using mitogenic agents which act intracellularly, the normal early increase in cell size with A23187- and PMA-stimulated lymphocytes was not affected by inhibitory leprosy sera or serum IgG, but all subsequent steps in the cell cycle were suppressed; although the inhibition of proliferation in PMA-stimulated cultures was incomplete. The mechanism of action of the inhibitory sera and derived IgG, although acting through a cell surface antigen, appears to interfere with a fundamental process in activation since the effect was seen with all of the diverse stimuli examined in this study.

Measurement of phagocyte chemiluminescence using a microtitre plate luminometer.

The use of chemiluminescence techniques to study the interaction between bacteria and phagocytes has been useful for examining the extent to which serum factors, such as opsonins, are important in internalization of the organisms and the response of the cell to phagocytosed bacteria. However, such methods have been limited by the number of experiments which can be performed at one time using most commercial luminometers. However, the recent introduction of the Amerlite microtitre plate luminometer allows the measurement of chemiluminescence responses in 96-well microtitre plates. Using this instrument, lucigenin-enhanced chemiluminescence can be detected from as few as 5000 cells (polymorphonuclear leukocytes or monocytes) per well with a 1:10 ratio of cells to zymosan particles opsonized with 10% serum. The opsonic capacity of up to 100 sera can be measured in triplicate wells in a single experiment using four microtitre plates and polymorphonuclear leukocytes prepared from less than 40 ml freshly obtained venous blood. We are currently using this technique to investigate the effect of serum opsonins on the interaction between normal human polymorphonuclear leukocytes and monocytes with mycobacteria of three species (Mycobacterium leprae, M. tuberculosis, and M. avium-intracellulare). Other possible applications of this method are discussed.