Lymphoproliferative responses of leprosy patients and healthy controls to nitrocellulose-bound M. leprae antigens.

The lymphoproliferative responses of 51 leprosy patients and 11 healthy contacts were analyzed using the nitrocellulose-bound specific antigen fractions from the cell-free extract of Mycobacterium leprae. The main proliferation-inducing fraction for peripheral blood mononuclear cells of the healthy contacts was found to be the Fraction II, bearing antigens in the range of 66-45 kDa. However, this fraction failed to induce lymphoproliferation in the leprosy patients, unlike healthy contacts (p < 0.032). The number of responders as well as the strength of the responses to 66-45 kDa proteins were found to be low in the leprosy patients compared to the healthy contacts. Further, preliminary analysis with the subfractions of Fraction II produced a similar pattern, suggesting that the immune response to the antigens in the range of 66-45 kDa M. leprae proteins remains suppressed in subjects with clinical signs and symptoms of the disease.

Lymphoproliferative responses of leprosy patients and healthy controls to nitrocellulose-bound M. leprae antigens

The lymphoproliferative responses of 51 leprosy patients and 11 healthy contacts were analyzed using the nitrocellulose-bound specific antigen fractions from the cell-free extract of Mycobacterium leprae. The main proliferation-inducing fraction for peripheral blood mononuclear cells of the healthy contacts was found to be the Fraction II, bearing antigens in the range of 66-45 kDa. However, this fraction failed to induce lymphoproliferation in the leprosy patients, unlike healthy contacts (p < 0.032). The number of responders as well as the strength of the responses to 66-45 kDa proteins were found to be low in the leprosy patients compared to the healthy contacts. Further, preliminary analysis with the subfractions of Fraction II produced a similar pattern, suggesting that the immune response to the antigens in the range of 66-45 kDa M. leprae proteins remains suppressed in subjects with clinical signs and symptoms of the disease.

Genetic variations at the T cell receptor gamma locus in circulating peripheral blood mononuclear cells of clinically categorised leprosy patients.

The allelic polymorphisms at exon 3 and exon 2 of the T cell receptor (TCR) C gamma 2 (TRGC2) gene, generating 18-kb and 5.4-kb HindIII fragments, respectively, were found to be more frequent in multibacillary leprosy patients than in the controls (P < 0.005 and P < 0.001, respectively) when screened with the IDP2.11 probe. The frequencies of heterozygotes for the 18-kb allele and homozygotes for the 5.4-kb allele were found to be significantly higher in the multibacillary patients than in the controls (P < 0.001). Interestingly, the 8.0-kb allele, originating from the triplication of exon 2 of C gamma 2, was observed exclusively in the paucibacillary leprosy patients. Further, when DNA samples were screened with the pH60 probe for the HindIII RFLP at the TCR J gamma 2 (TRGJ2) gene segment, the 2.1-kb allele was again more prevalent in leprosy patients with the multibacillary form of the disease than in the paucibacillary patients and the controls (P < 0.025). The frequency of homozygotes for the 2.1-kb allele was also significantly higher in the multibacillary patients than in the paucibacillary patients (P < 0.010) and the controls (P < 0.025). A significant difference was observed in the frequencies of detectable rearrangements involving the V gamma 7/8 and V gamma 9 gene segments at the gamma locus between circulating peripheral blood mononuclear cells of the multibacillary leprosy patients and the controls. These rearrangements were detected less frequently in the multibacillary patients (P < 0.001 for V gamma 7/8 and P < 0.005 for V gamma 9).

Association of polymorphism at COL3A and CTLA4 loci on chromosome 2q31-33 with the clinical phenotype and in-vitro CMI status in healthy and leprosy subjects: a preliminary study.

Two genetic loci, viz. COL3A and CTLA4, located within the chromosome 2q31-33 region in the vicinity of the proposed syntenic site of the mouse "Bcg" locus were genotyped by the polymerase chain reaction in leprosy patients and healthy individuals. All the subjects studied were assessed as in-vitro responders/non-responders to mycobacterial antigens. Simple sequence length polymorphism analysis revealed five (236 to 312 bp) and eight (84 to 120 bp) allelomorphs for COL3A and CTLA4, respectively. Our preliminary analysis showed a significant association between the 250-bp COL3A allelomorph in the homozygous condition and the multibacillary form of leprosy (P < 0.05: relative risk = 5.5). Another allelic (312 bp) variant of COL3A was significantly correlated with non-responsiveness to M. leprae antigens in vitro (P < 0.01). The 104-bp allelomorph of CTLA4 was not observed in any of the 25 cases of leprosy. This absence was statistically significant (P < 0.05) when compared with normal healthy controls and depicted a high relative risk (RR = 25.83). An additional observation of the predominance of a unique 84-bp CTLA4/CTLA4-like allelomorph was observed in the Indian subjects studied.

Quantitative assessment of tuftsin receptor expression and second messenger during in vitro differentiation of peripheral blood derived monocytes of leprosy patients.

Tuftsin, a tetrapeptide (Thr-Lys-Pro-Arg) is known to potentiate the immunogenic activity of antigen-fed macrophages. The present study describes the mechanism of action of tuftsin in leprosy patients throughout the spectrum of the disease in vitro as a function of culture age in terms of (A) involvement of second messengers cAMP, cGMP and [Ca2+]i and (B) number of tuftsin binding sites/and their relative affinities on the monocytes/macrophages. There is apparently no direct involvement of either cAMP or cGMP while comparing the stimulated and unstimulated cultures during in vitro differentiation of monocytes (days 1, 3 and 7) or with the spectrum of the disease. Inhibition of superoxide anion release either by verapamil or with Quin 2 clearly demonstrated the involvement of [Ca2+]i as a second messenger during activation of monocytes/macrophages with tuftsin. Scatchard analysis of radiolabelled tuftsin binding data showed only one type of tuftsin receptor (low affinity) on BL/ LL monocytes/macrophages and normal and BT/TT cultures showed a gradual change in receptor number and affinities (low to high) with the maturation of monocytes to macrophages in contrast to BL/LL groups which displayed significantly less number of receptors. This study elicits a model which depicts that the biological responses/metabolic functions of early monocytes of normal and BT/TT gradually increase with the age of the culture till day 3 and tapers off thereafter in the older (day 7) cultures, whereas the monocytes/macrophages of BL/LL group are metabolically active only on day 1. The present study thereby implies that the clearance of leprosy bacilli from lepromatous leprosy lesions as a consequence of local or systemic immunotherapy (in the present study, the macrophage modulation by tuftsin) depends on the influx of new competent macrophages, rather than the local activation of resident lepromatous macrophages.

Interdependence between degree of porphyrin excess and disease severity in congenital erythropoietic porphyria (Günther's disease).

Various clinical and biochemical observations point to a relationship between degree of disease expression and metabolic disturbance in autosomal recessive congenital erythropoietic porphyria (Günther's disease). Although the clinical manifestations have been well described since Günther's fundamental observations, an interdependence between disease severity and porphyrin excess has yet to be elucidated. We investigated porphyrin metabolism in nine Indian patients suffering from the characteristic clinical symptoms: skin photosensitivity, red-colored urine as a sign of extremely elevated porphyrinuria and mild to severe hemolytic anemia. Porphyrins in urine, feces and blood were analysed by HPTLC and HPLC in conjunction with spectrophotometry and spectrofluorometry. Uroporphyrinogen III synthase activities in red blood cells were determined using a coupled-enzyme assay. Biochemical studies revealed varying degrees of porphyrinuria with total urinary porphyrins between 23 and 102 mumol/24 h (normal < 0.2 mumol/24 h) and uroporphyrin predominance. Urinary and fecal coproporphyrin isomer I were markedly elevated to 87-97% and 81-93% (normal < 31%, < 75%), respectively. Overproduction of porphyrins led to a considerable porphyrinemia with mainly copro- and protoporphyrin. A hitherto undescribed fecal porphyrin pattern with increased protoporphyrin levels was found in three patients. This atypical finding was probably related to severe hemolysis since protoporphyrin can be excreted only via the liver with bile in the feces. High porphyrin levels in urine, feces and blood were associated with worse cutaneous symptoms. Activities of uroporphyrinogen III synthase in red blood cell lysates were decreased to between 9% and 30% of controls. Patients showed increased porphobilinogen deaminase activities, up to 190% of control. Deficiency of uroporphyrinogen III synthase activity was reflected by inversion of the relationship between and isomer III leading to dominance of isomer I. Elevation of porphobilinogen deaminase activities is related to hemolysis and, additionally, to regulatory compensation for the enzyme deficiency. Variations in both the severity of photosensitivity and the enhancement of porphyrin production and excretion indicate the molecular heterogeneity of this disease. These findings suggest a close relationship between the metabolic disturbance reflected by porphyrin excess and the severity of disease expression.

Release of reactive nitrogen intermediates from the peripheral blood-derived monocytes/macrophages of leprosy patients stimulated in vitro by tuftsin.

The production of reactive nitrogen intermediates (RNI) by macrophages is critical to host defence, particularly for exerting the bactericidal and tumoricidal properties. Nitric oxide (NO) were measured in the peripheral blood-derived monocytes/macrophages of normal and leprosy patients (BT/TT and BL/LL) in the presence and absence of 'tuftsin' as a function of in vitro culture age (on 1, 3, 7 days). Macrophages from both groups of leprosy patients were able to produce NO during the unstimulated state but only BL/LL macrophages could be activated by tuftsin to produce significantly high levels of NO. This increase was highest on day 1, then gradually decreased with in vitro culture age. Surprisingly, tuftsin was unable to enhance the NO production in normal macrophages above the basal level. Further, normal and BT/TT macrophages had only Cu-Zn derived superoxide dismutase (SOD) activity whereas BL/LL cultures has Cu-Zn and Mn derived SOD activity. These studies indicate that in BL/LL cultures: a, apart from tuftsin, some additional signal is required to activate nitric oxide synthase (NOS) gene for NO production; and b, Mn-SOD produced by Mycobacterium leprae is playing a defensive role against toxic-free radicals. The final outcome of this mechanism is the survival of M. leprae inside the macrophages.

Dual porphyria of coexisting variegata and cutanea tarda.

While porphyria cutanea tarda and porphyria variegata are independent diseases, we report on seven rare cases with a coincidence of these two different porphyrias in one individuum. The mutual clinical symptom was a cutaneous photosensitivity, which is a major symptom in porphyria cutanea tarda and a facultative one in porphyria variegata. Additionally, five patients had also experienced episodes of acute abdominal pain, which were in three cases accompanied by neurological symptoms, thus offering evidence for an acute hepatic porphyria, such as porphyria variegata. Determination of urinary porphyrin metabolites revealed a porphyria cutanea tarda-like excretion pattern with an elevation of uroporphyrin (mean 1134 nmol/24 h, range 563-4052, normal < or = 30) and heptacarboxyporphyrin (mean 389 nmol/24 h, range 64-830, normal < or = 4). In all patients, however, urinary coproporphyrin was also increased, reaching levels too high for porphyria cutanea tarda but typical for porphyria variegata (mean 1788 nmol/24 h, range 142-4168, normal < or = 120). Fecal porphyrin excretion also resembled the variegate-type with a high concentration especially of protoporphyrin (mean 628 nmol/g dry weight, range 401-1018, normal < or = 151), accompanied by an increase of coproporphyrin (mean 194 nmol/g dry weight, range 75-409, normal < or = 37). The urinary porphyrin precursors 5-aminolaevulinic acid and porphobilinogen were markedly elevated only in one patient, who was in an acute porphyric phase at the time of investigation. The activity of uroporphyrinogen decarboxylase in erythrocytes was considerably decreased in six of our cases (33-64%) and slightly diminished in the other one (83% of normal activity).(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of peripheral blood derived monocytes/macrophages from leprosy patients using 'tuftsin' for production of reactive oxygen intermediates.

Phagocytic cells respond to a variety of membrane stimulants by producing reactive oxygen intermediates (ROI), i.e. O2-, H2O2 and OH.metabolites. Plasma membrane activation is associated with superoxide generating NADPH oxidase, thereby causing the production of these toxic species. Stimulation of phagocytic cells also results in activation of purine catabolism, which directs the metabolic flux through xanthine oxidase to produce the superoxide anion. We previously observed that BL/LL macrophages (M phi) exhibited a premature inability to undergo tuftsin stimulated phagocytosis and microbicidal activity. The present study was undertaken to measure ROI levels in the absence and presence of 'tuftsin' pulsing as a function of in vitro culture age and also correlated these levels with adenosine deaminase (ADA) activity. The latter is known to be a contributor of O2- generation and is also involved in the maturation of the monocyte/macrophage system. The behaviour of normal and tuberculoid monocytes/macrophages were more or less the same, either in the presence or absence of tuftsin, i.e. they showed a progressive increase in ROI production until day 3, then tapered off in older cultures by day 7. In contrast, after day 1, the lepromatous macrophages were unable to undergo tuftsin mediated stimulation for the production of ROI and ADA activity. These findings indicate a defective M phi function in lepromatous patients towards tuftsin pulsing, thereby supporting our earlier observations. Thus BL/LL M phi behaved as if they were aged after 1 day of in vitro culture, which may account for an inability to handle Mycobacterium leprae for efficient killing.

Enzyme immunoassay of phagocytosis stimulating tetrapeptide "tuftsin" in normal and leprosy sera.

The serum concentrations of the phagocytosis stimulating the tetrapeptide, tuftsin, were determined by competitive enzyme immunoassay in borderline tuberculoid/tuberculoid (BT/TT, 16 cases), borderline lepromatous/lepromatous (BL/LL, 16 cases), and in healthy controls (20 cases). Using checkerboard titration, 10 ng/well of diphtheria toxoid-p-aminophenylacetyl tuftsin (DTPT) conjugate when incubated with tuftsin antisera at 1:15,000 dilution with a preincubation time of 60 min with the competitor (tuftsin) followed by a further 60-min incubation onto the DTPT-coated wells gave consistent results with a sensitivity of 5 ng/well tuftsin. The mean serum tuftsin concentration was significantly lower in BL/LL patients (134.42 +/- 48.7 ng/ml, p less than 0.01) than in healthy controls (262.86 +/- 59.8 ng/ml), while BT/TT sera (210.94 +/- 75.5 ng/ml) showed slightly decreased levels than did normals, which was not statistically significant. The mean serum IgG levels in BL/LL and BT/TT patients (37.26 +/- 10.99 mg/ml; 28.08 +/- 6.57 mg/ml, respectively) showed significantly (p less than 0.001) higher concentrations than did healthy controls (12.3 +/- 3.6 mg/ml). These observations on the serum concentrations of tuftsin and IgG in leprosy individuals suggest that there is splenic dysfunction in BL/LL patients in terms of the processing of leukokinin to release the free, active molecule tuftsin.

Modulation of human lepromatous monocyte-macrophage functions in vitro by tuftsin.

Human peripheral blood monocytes/macrophages derived from normal donors, patients of tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) were assayed for stimulated phagocytic responses to the potent macrophage stimulator "Tuftsin" (NH2-Thr-Lys-Pro-Arg-OH) after varying periods (6 h to 14 days) of culture in vitro. The assays consisted of visual scoring of ingested Mycobacterium leprae and radiometric measurement of ingested 14C-acetate labelled Staphylococcus aureus and Mycobacterium tuberculosis (H37Ra). While normal and BT/TT macrophages showed a progressively increasing ability for tuftsin-stimulated phagocytosis with increasing age of culture in vitro, BL/LL macrophages showed the opposite response so that 14-day cultures were refractory to a stimulatory dose of up to 7.0 microM (10 to 20 times the optimal dose for normal and BT/TT macrophages). The 14-day BL/LL macrophage cultures were, however, responsive to 35 microM tuftsin (100 times the optimal dose for normal macrophages). Analysis of the dose-response curves also indicates that BT/TT cultures despite exhibiting an apparent similarity to normal macrophages demonstrate a rightward shift for a maximal stimulated phagocytosis. Finally SEM photo-micrographs of 14-day macrophage cultures of the three groups revealed that while normal and BT/TT cultures demonstrated an increase in membrane ruffling and filopodia on stimulation with 0.8 microM tuftsin, BL/LL cultures exhibited none of the features associated with stimulation. From the above findings, we conclude that lepromatous macrophages may display an aberrant differentiation profile leading to a terminal state of unresponsiveness and that the defect may possibly lie at the level of tuftsin receptor expression or transmembrane signal transduction.

Effect of tuftsin stimulation on the microbicidal activity exerted by blood monocyte-macrophages of leprosy patients.

The ability of blood monocyte/macrophages from normal donors, tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) patients to exert enhanced microbicidal activity was assayed after stimulating with 0.8 microM tuftsin, as a function of the duration of cultures in vitro. Normal and BT/TT macrophage cultures showed a statistically significant increase in microbicidal activity against Staphylococcus aureus at all ages of culture (6 h to 14 days), though the overall magnitude of the enhancement shows a decrease with increasing culture age in the same populations. However, 14-day old BL/LL macrophage cultures were unable to undergo tuftsin-mediated stimulation of microbicidal activity against S. aureus and even, fresh 6 h-old cultures exhibited a tuftsin-stimulated response profile similar to 14-day old normal and BT/TT cultures. Also, 7 and 14-day cultures of normal, BT/TT and BL/LL macrophages were unable to inhibit/kill intracellular Mycobacterium leprae after a single stimulation with 0.8 microM tuftsin. However, serial, daily stimulation with 0.8 microM tuftsin resulted in 77-140% inhibition of 3H-thymidine uptake by the 12th day of cultures in vitro in the three groups. These results suggest that BL/LL macrophages exhibit a premature inability to undergo tuftsin stimulated microbicidal activity, which may possibly be reversed by serial dosage of tuftsin.