Phenotypic determination of T-lymphocytes responding to chemotactic stimulation from fMLP,IL-8, human IL-10, and epidermal lymphocyte chemotactic factor

Summary Human T lymphocytes were collected after they had migrated towards N-formyl-methionyl-leukyl-phenylalanine (fMLP), rIL-8, human IL-10 (hIL-10), and epidermal lymphocyte chemotactic factor (ELCF). They were stained for determination of their phenotype by FACS analysis using anti-CD4, -CD8, -CD18, -CD45R0 and OPD4 antibodies. Human IL-10 increased the percentage of CD8⁺ T lymphocytes in the migrating cell population by 152% compared with cells migrating towards the medium and decreased the number of CD4⁺ T lymphocytes by 79%. ELCF increased the number of CD4⁺ T lymphocytes by 18%, and the number of CD45R0⁺ T lymphocytes by 52%, while the number of CD8⁺ T lymphocytes was decreased by 20%. rIL-8 increased the number of CD4⁺ T lymphocytes and decreased the CD8⁺ T lymphocytes. The distribution of the different subpopulations of T lymphocytes was not changed significantly by fMLP. The observed changes in the phenotypes did not occur when incubating T lymphocytes with the chemotaxins. Our observations demonstrate that individual chemotactic factors will attract specific subsets of T lymphocytes. They may help to explain the predominance of memory T lymphocytes (CD4R0⁺, CD4⁺) in allergic contact dermatitis and certain other skin diseases. They also confirm the results of a recent study, that showed hIL-10 to be selectively chemotactic for CD8⁺ T lymphocytes.     

Circulating CD4+ CD25 high FoxP3+ T cells vary in different clinical forms of leprosy

Background CD4⁺ CD25^highFoxP3⁺ regulatory T cells (T‐regs) were reported to increase in chronic infections. We aimed at studying their frequency in leprosy to investigate their role during Mycobacterium leprae infection. Methods Using flow cytometry, the frequency and FoxP3 expression of circulating T‐regs was assessed in 38 leprosy patients and 38 healthy controls. Patients were divided into; group I tuberculoid (TT), group II borderline cases [borderline tuberculoid (BT), borderline (BB), and borderline lepromatous (BL)], group III lepromatous (LL), and group IV erythema nodosum leprosum (ENL). Results Mean T‐regs% and FoxP3 expression were significantly elevated in patients (particularly TT) compared to controls (3.8 ± 2.5% vs. 2.5 ± 0.8% and 78.8 ± 56.2% vs. 55.8 ± 15.7%, respectively) (P < 0.05). Comparing the four disease groups, T‐regs% was significantly different (median 5.3% in group I, 3.4% in group II, 2.8% in group III, and 1.2% in group IV; P = 0.005). FoxP3% on T‐regs was not significantly different between them [median 71.5% in TT, 62.3% in borderline categories, 67.75% in LL, and 85.75% in ENL; P = 0.149). Notably FoxP3 expression was significantly higher in ENL than controls (P = 0.011). Conclusion The frequency and suppressive marker of circulating T‐regs are elevated in TT patients. Patients with LL and ENL express significantly lower frequency of T‐regs and higher FoxP3 expression (in ENL), consistent with disease progression and immune hyper‐activation in these disease categories. Thus, rather than being detrimental to immunity, intact T‐regs activity may be beneficial to leprosy patients.     

LEPROMATOUS AND TUBERCULOID LEPROSY: CLINICAL PRESENTATION AND CYTOKINE RESPONSES

Objective. This study analyzes the major clinical characteristics of patients with active leprosy in relation to the in vitro immune response to the T- lymphocyte activator anti-CD3. Methods. Thirty-eight patients with an established diagnosis of leprosy were classified according to the Ridley and Jopling table. Peripheral blood mononuclear cells from both lepromatous leprosy (LL) and tuberculoid leprosy (TL) patients and healthy controls were used to evaluate lymphocyte proliferation; immunoenzymatic assays were used to evaluate cytokine production (IL-I, IL-2, IL-4, IL-6, IL-10, IFN-gamma). Results. Peripheral blood mononuclear cells from both LL and TL patients displayed blastogenic responses to anti- CD3. The cytokines IL-1β, IL-6, IL-10, and IFN-γ were detected in culture supernatants. Endogenous production of IL-1ß was significantly higher in cell cultures from patients with the lepromatous form of the disease compared to those with tuberculoid leprosy. Production of IL-6 in response to anti CD3 was observed in a significantly higher proportion of LL than TL patients (P = 0.0025). Gamma-interferon production did not differ between TL and LL, but a direct correlation was observed between time of multidrug treatment and IFN production in vitro (P = 0.016). Interleukin-10 was detected in culture supernatants of lymphocytes activated by anti-CD3 from both patient groups, but not from healthy controls. Conclusions. The findings of this study suggest that patients with the two distinct forms of leprosy are capable of responding to a polyclonal T-lymphocyte stimulus such as anti-CD3 and provide evidence suggestive of alterations in the immune responses mediated by cytokines that may contribute to the spectrum of disease and response to treatment.     

Serum Th17 cytokines in leprosy: correlation with circulating CD4+ CD25highFoxP3+ T-regs cells,as well as down regulatory cytokines

Leprosy is not only a bacteriological disease but also an immunological disease, in which T helper17 and CD4⁺ CD25^highFoxP3⁺ regulatory T cells (T-regs), among others, may play a role. We aimed to evaluate serum levels of interleukin (IL)-17, IL-22 (Th17 cytokines), IL-10 and transforming growth factor (TGF)-β (down regulatory cytokines) in 43 untreated leprosy patients and 40 controls by enzyme-linked immunosorbent assay, and to assess circulating CD4⁺ CD25^highFoxP3⁺T-regs in patients using flow cytometry. Patients were grouped into tuberculoid, pure neural, borderline, lepromatous, type 1 reactional leprosy, and erythema nodosum leprosum. IL-10 and TGF-β were significantly higher in patients as compared to controls (p < 0.001), while IL-17, but not IL-22, was significantly lower (p < 0.001), with no significant difference comparing patients’ subgroups. Significantly higher CD4⁺ CD25^highFoxP3⁺T-regs levels was detected in tuberculoid, type 1 reaction and pure neural leprosy, while the lowest levels in erythema nodosum leprosum (p < 0.001). TregsFoxP3 expression% was significantly lower in pure neural leprosy than other patients’ subgroups (p < 0.05). T-regs/T-effs was lowest in erythema nodosum leprosum (p < 0.05). TGF-β correlated negatively with TregsFoxP3 expression% and T-effs% (p = 0.009 and 0.018 respectively). Leprosy is associated with defective IL-17 and overproduction of IL-10 and TGF-β. Tuberculoid, type 1 reaction and pure neural leprosy express significantly higher circulating T-regs, consistent with effector immune mechanisms activation, but with lower TregsFoxP3 expression (in pure neural leprosy). Erythema nodosum leprosum is characterized by deficient T-regs and increased TregsFoxP3 expression%. The present study pinpointed a potential role of Th17, CD4⁺ CD25^highFoxP3⁺T-regs, and probably CD4⁺ CD25⁺IL-10⁺ T regulatory cells 1 (Tr1), and Th3 in leprosy.     

Increased incidence of cytoplasmic ANCA (cANCA) and other autoantibodies in leprosy patients from western India

The prevalence of various autoantibodies was studied in 75 leprosy patients comprising eight patients with lepromatous leprosy (LL), 36 patients with borderline lepromatous leprosy (BL) and 31 patients with borderline tuberculoid leprosy (BT), along with 100 normal controls. Certain autoantibodies such as anti-nuclear antibodies (ANA), anti-single stranded DNA (anti-ssDNA) and anti-neutrophil cytoplasmic antibodies (ANCA) were raised among leprosy patients. When ANCA specificities to anti-myeloperoxidase (anti-MPO), anti-proteinase3 (anti-PR3) and anti-lactoferrin (anti-LF) were studied, it was found that the patterns of immunofluorescence such as perinuclear (p-ANCA), cytoplasmic (c-ANCA) and atypical (X-ANCA) and specificity by ELISA to anti-MPO, anti-PR3 and anti-LF varied in the LL, BL and BT groups. However, a higher amount of c-ANCA was observed in 62.5% of leprosy cases, while the incidences of p-ANCA and X-ANCA were lower. The LL group showed a higher incidence of autoantibodies as compared with the BL and BT groups, along with a male preponderance for autoantibody development. Some unusual antibody profiles such as 'X'-ANCA were also observed. The study suggests that autoantibody formation could be quite prevalent and also variable in the spectrum of leprosy cases, and there seems to be a serological overlap among leprosy and autoimmune disease, which could have pathogenetic importance in the leprosy patients developing complications.     

Diagnosing leprosy: revisiting the role of the slit-skin smear with critical analysis of the applicability of polymerase chain reaction in diagnosis

Background Diagnosing leprosy is challenging, especially in early‐stage cases, and the need for a sensitive diagnostic tool is urgent. Polymerase chain reaction (PCR) holds promise as a simple and sensitive diagnostic tool, but its usefulness in the Indian context requires further evaluation. Slit‐skin smear (SSS) remains the conventional method of leprosy detection. Hence, this study was undertaken to evaluate and compare the diagnostic efficacy of PCR versus that of SSS. Methods Punch biopsy of skin and SSS were obtained from the active margins of lesions. Cases were clinically grouped according to whether they were multibacillary (MB) or paucibacillary (PB) and classified into tuberculoid (TT), borderline tuberculoid (BT), borderline lepromatous (BL), lepromatous (LL), histoid, and indeterminate groups after clinicopathological correlation. DNA was extracted from biopsy specimens, and multiplex PCR was carried out incorporating primers intended for the amplification of a specific 372‐bp fragment of a repetitive sequence of Mycobacterium leprae DNA. Results Among 164 patients, PCR was positive in 82.3%. The sensitivity of PCR was significantly greater (P < 0.0001) than that of SSS in both the MB (85.9% vs. 59.8%) and PB (75.4% vs. 1.8%) subgroups; the difference in sensitivity in the PB subgroup is remarkable. Positivity by PCR and SSS was found in 100% of LL and histoid leprosy, but PCR had significantly greater (P < 0.0001) positivity in BT leprosy and was of definite increased value in indeterminate and TT leprosy. Conclusions Polymerase chain reaction had higher sensitivity compared with SSS, especially in diagnostically challenging and PB cases. Thus, the use of this costly but sensitive tool should be restricted to this subgroup, because SSS is sufficiently sensitive in the diagnosis of LL and histoid leprosy.     

Alpha-1-antitrypsin in leprosy

Estimation of Alpha-1-antitrypsin (AAT) levels was carried out in 52 patients of various types of leprosy. Fifty age and sex matched healthy individuals served as controls. The mean level of AAT in controls was 290.12 +/- 59.56 mg/dl. In patients of tuberculoid leprosy (TT), borderline tuberculoid leprosy (BT) and borderline leprosy (BB), the AAT levels were found to be 284 +/- 47.03, 314.37 +/- 31.56 and 324.44 +/- 32.05 mg/dl respectively. These were statistically insignificantly raised when compared with controls. In borderline lepromatous leprosy (BL), lepromatous leprosy without erythema nodosum leprosum (LL without ENL) and in LL with ENL there was a statistically significant rise in AAT levels. The maximum levels of AAT were observed in patients of LL with ENL (mean 500.8 +/- 93.44 mg/dl. P less than 0.001).     

HLA和麻风相关的研究Ⅱ.L型麻风与HLA-DR2相关

本文对69名江苏汉族麻风病人(L型32名.T型37名》进行了HLA-DR分型,弃分别与65名健康对照相比较.观察到全部病人组和L型病人组DR2抗原频率显著增高,校正后仍有显著性(两组分别为X²=9.16,P=0.003,Pc=0.024,RR=3.15;X²=11.69,P=0.0006,Pc=0:005).T型缪观察到DR:低度增加一(频率差为0.163),但无显著意叉.恨据Porta-McHugh公式,按常巢色体隐性模型(A.Rmodel)用HLA-SA-B程序计算216个HLA-ADR,HLA-B,DR单语型筒假设的麻风易感基因.三位点连锁不平衡参数相对值,发现了四个相对易感麻风的单倍型,All-DR2为相对易感L型麻风的单倍型A9-DR2,All-DRs,Bwso-DR3是相对易感T型麻风的单倍型.本文结果进一步提示;假设的麻风易威基因是同HLA区域相连锁的,向接说明疾病易感基因可能在DR位点侧两型麻风均司能呈多抗原弱相关.两型麻风具有不同的遗传背景一可能是尸组异质性疾病.     

Thalidomide's effectiveness in erythema nodosum leprosum is associated with a decrease in CD4+ cells in the peripheral blood.

Thalidomide is well documented as being an effective drug in the treatment of erythema nodosum leprosum (ENL). The mechanism of action of thalidomide in ENL as well as the pathogenesis of ENL are yet to be fully determined. Lepromatous leprosy patients experiencing ENL have been reported to have an increase in the ratio of CD4+ to CD8+ cells in their blood and ENL skin lesions. Thalidomide has been shown to cause a decrease in the ratio of CD4+ to CD8+ lymphocytes in the blood of healthy males. This decrease was due to a significant reduction in the numbers of Cd4+ lymphocytes and an apparent increase in the numbers of CD8+ lymphocytes. In this study, thalidomide's effectiveness in halting chronic ENL and arresting a relapse into ENL was consistently associated with a decrease in the numbers of CD4+ lymphocytes in the blood of 2 male lepromatous leprosy patients.     

Erythrocyte superoxide dismutase, catalase activities and hydrogen peroxide induced lipid peroxidation in leprosy

OBJECTIVES: To assess erythrocyte superoxide dismutase (SOD) and catalase (CAT) activities and hydrogen peroxide induced lipid peroxidation in leprosy. DESIGN: One hundred leprosy patients and 50 normal healthy controls were studied for the parameters. The data was analysed by grouping the patients into Ridley-Jopling (RJ) types [Tuberculoid leprosy (TT, n = 22), Borderline tuberculoid leprosy (BT, n = 28), Borderline leprosy (BB, n = 13), Borderline lepromatous leprosy (BL, n = 16) and Lepromatous leprosy (LL, n = 21)] and into different levels of Bacteriological Index (BI) [bacteriologically negative (n = 32), BI = 0.1-1 (n = 22), BI = 1.1-2 (n = 16), BI = 2.1-3 (n = 14), BI = 3.1-4 (n = 10) and BI = 4.1-6 (n = 06)]. RESULTS: The induced peroxidation was significantly high and the enzyme activities were significantly low in leprosy (total patients) as compared to controls. A progressive increase in peroxidation was detected along the leprosy spectrum from TT to LL and the increase was significant in BB, BL and LL groups as compared to controls. Induced peroxidation in LL group as compared to TT, BT and BB and in the BL group as compared to TT and BT were significantly different. A concomitant progressive decline in enzyme activity was detected along the leprosy spectrum from TT to LL. The SOD activity in BB, BL and LL and the CAT activity in BL and LL were significantly low as compared to controls. SOD activity in BB, BL and LL groups as compared to TT and in the LL group as compared to BT were significantly different. A progressive trend of increasing peroxidation and decreasing SOD and CAT activity were also detected along the leprosy groups with advancing level of BI. Induced peroxidation and SOD activity were significantly different in bacteriologically positive groups as compared to controls and in the BI levels 1.1-2, 2.1-3, 3.1-4 and 4.1-6 as compared to bacteriologically negative group. The peroxidation was significantly different in BI levels 2.1-3, 3.1-4 and 4.1-6 as compared to BI level 0.1-1. The CAT activity was significantly different in BI levels 2.1-3, 3.1-4 and 4.1-6 as compared to controls. CONCLUSION: The study findings suggest oxidative stressful state associated with reduced antioxidant defence potential in erythrocytes of leprosy patients. The study implicates association of erythrocyte oxidative stress with bacterial load and type of leprosy.     

Correlation of serum lipids and lipoproteins in leprosy.

Serum lipids and lipoproteins were assessed in sixty leprosy and forty age and sex matched healthy controls. The study subjects included cases of LL with reactions, LL without reactions, BL with reactions, BL without reactions, BT and TT types of leprosy. The levels of serum phospholipids, triglycerides, total cholesterol, LDL and VLDL fractions were significantly decreased in leprosy patients compared to control subjects. The levels of serum HDL cholesterol and HDL fraction were significantly elevated in leprosy patients. Maximum elevation in serum HDL cholesterol level and HDL fraction and maximum reduction in the levels of serum phospholipids, triglycerides, total cholesterol and LDL and VLDL fractions were observed in lepromatous leprosy (LL) patients with reactions.     

Dermatoglyphics in leprosy (II Metric analysis of palms)

A group of 100 leprosy patients consisting of 50 lepromatous (BL/LL) and 50 tuberculoid (BT/TT) were investigated for metric analysis of the patterns present on their palms. Hundred normal persons were also selected from the families of patients to serve as controls. BT/TT patients and controls did not show any significant difference in their palmar patterns. On the other hand, significant differences were observed in the patterns between BL/LL patients and controls.     

Quality of life in patients with lepromatous leprosy in China

Background Leprosy has an impact on patients’ quality of life (QoL). However, there has been no study specifically on the impact of the severest type of leprosy‐lepromatous leprosy on QoL. Objective To describe the use of Dermatology Life Quality Index (DLQI) among patients with lepromatous leprosy in China. Methods Sixty‐four inpatients with lepromatous leprosy of Shangluo hospital and Hanzhong hospital and sixty‐four controls (healthy volunteers or patients with other dermatoses) matched for age and gender were asked to complete DLQI questionnaires from 2 September 2008 to 20 December 2008. Extensive data were collected besides DLQI, including demographic data and disease‐related characteristics. Absence or presence of disability among patients with lepromatous leprosy was evaluated at the same time. Results The overall mean DLQI score for lepromatous leprosy (18.78) was higher than that for control (9.00) (P < 0.001). Patients with lepromatous leprosy scored significantly higher for all items (P < 0.001) except Q4 (clothes choice). Controls scored significantly lower for all domains of DLQI. Scores of LL increased markedly with increasing clinical severity, but were not associated with educational level, gender, age and disease duration. The inter‐item correlation averaged 0.240 and Cronbach’s alpha was 0.759, indicating high internal consistency. Conclusions This is the first exclusive study to attempt to measure the impact of lepromatous leprosy on QoL. Lepromatous leprosy has a severe impact on QoL.     

透明质酸及其受体CD44在麻风患者愈后表皮中的表达

目的研究透明质酸及其受体CD44在麻风患者皮损愈后者表皮的表达是否发生改变。方法用免疫组化方法比较透明质酸及其受体CD44在不同类型麻风患者愈后及正常表皮中的表达。结果与正常表皮比较,透明质酸在LL、BL及BT型麻风患者皮损愈后表皮的表达明显增强;而在TT型表皮的表达没有明显的改变;在BB型基底层的表达也轻微减弱。CD44在BB,BT及TT类型麻风患者愈后表皮的表达均增强,尤其是TT型。而在LL型的表达则轻微减弱。CD44在BL型的表达没有明显改变。结论麻风患者愈后表皮透明质酸及CD44的表达均有改变。     

Dermatoglyphics in leprosy: (III) Creases of palm

Palmar configurations of triradii and creases of 100 leprosy patients [50 lepromatous (BL/LL) and 50 tuberculoid (BT/LL)] were compared with those of 100 normal persons selected from families of these patients. The patterns of position of triradii were similar in controls and leprosy patients as such. But, the patterns in the two types of leprosy patients were different. As for palmar creases patterns, there was significant difference between those of controls and patients, double radial base crease occurring more often in patients. However, the differences between the two types of patients were not statistically significant.     

IMMUNOHISTOCHEMISTRY IN HISTOID LEPROSY

Background. Histoid leprosy is a rare form of multibacillary leprosy as the result of secondary or even primary resistance to dapsone. The etiopathogenesis has not been clarified up to now. Methods. An immunohistochemical study was carried out for the expression of various markers on epidermal and dermal cell populations using sections of frozen skin specimens from 5 patients with histoid leprosy as compared to specimens from 7 tuberculoid and 7 lepromatous patients. Results. Dendritic epidermal cells, identified by monoclonal antibodies against CD1, hla-dr , CD45, and CD36, were found reduced in histoid leprosy as compared to both tuberculoid and lepromatous groups. A gradual reduction of keratinocytic hla-dr expression from tuberculoid to lepromatous to histoid leprosy was observed. The pattern of CD36, CD4, and CD8 expression of lymphomonocytic cells in the dermis of histoid lesions was similar to that of tuberculoid leprosy, but without the formation of an organized granuloma. CD45+ cells as well as activated lymphocytic cells, expressed by the activation immunophenotype (CD1, hla-dr , CD25, CD71, egf-r ) were found frequently in all groups. Conclusions. The in situ immunohistochemical findings support a modified hypersensitivity reaction of the cellular type that results in an inhibition of the lesional expansion, but not in the destruction of the bacilli within the histoid lesion.     

Evidence that regression in Keratoacanthoma is immunologically mediated: a comparison with squamous cell carcinoma

Recent research observations suggest that the keratoacanthoma (KA) is a form of resolving squamous cell carcinoma (SCC). The mechanism by which this resolution takes place has not been fully explored, although it may have an immunological basis. To investigate this, we compared 15 clinically and histologically diagnosed KAs and 15 SCCs with regard to cellular infiltrate and keratin expression. We found that KAs have significantly higher numbers of CD3⁺ and CD4⁺ cells invading their epidermal component than SCCs. The lymphocytes infiltrating KAs were more immunologically active, as greater numbers expressed the interleukin-2 receptor (IL-2R) than those in SCCs. It is of interest that CD36 was expressed by a significantly greater proportion of tumour cells within KAs than SCCs. This was also the case for the intercellular adhesion molecule ICAM-1, and the differentiation marker keratin 10. Overall, these findings suggest that KA regression is immunologically mediated, with activated (IL-2R⁺) CD4⁺ T lymphocytes and adhesion molecules playing a pivotal role in the immune response.     

Immunohistopathological spectrum in cutaneous tuberculosis

Granulomas of lupus vulgaris were characterized by a raised CD4⁺/CD8⁺ ratio, while in scrofuloderma this was significantly decreased. Tuberculosis verrucosa cutis showed intermediate changes. These immunological variations suggest that cutaneous tuberculosis forms a spectrum with strong cell-mediated immunity seen in lupus vulgaris, and weaker cell-mediated responses in scrofuloderma; tuberculosis verrucosa cutis is intermediate between the two.     

T regulatory cells (TREG)(TCD4+CD25+FOXP3+) distribution in the different clinical forms of leprosy and reactional states

BACKGROUND

Leprosy is characterized histologically by a spectrum of different granulomatous skin lesions, reflecting patients'' immune responses to Mycobacterium leprae. Although CD4+CD25+ FoxP3+ T regulatory cells are pivotal in the immuneregulation, presence, frequency, and distribution of Tregs in leprosy, its reactional states have been investigated in few studies.

OBJECTIVES

This study aimed to verify the frequency and distribution of regulatory T cells in different clinical forms and reactional states of leprosy.

METHODS

We performed an immunohistochemical study on 96 leprosy cases [Indeterminate (I): 9 patients; tuberculoid tuberculoid: 13 patients; borderline tuberculoid: 26 patients; borderline borderline: 3 patients; borderline lepromatous: 8 patients; lepromatous lepromatous: 27 patients; reversal reaction: 8 patients; and erythema nodosum leprosum: 2 patients].

RESULTS

FoxP3-positive cells were present in 100% of the cases with an average density of 2.82% of the infiltrate. Their distribution was not related to granulomatous structures or special locations. There was a statistically significant increment of FoxP3 expression in patients with leprosy reversal reactions when compared with patients presenting with type I leprosy (P= 0.0228); borderline tuberculoid leprosy (P = 0.0351) and lepromatous leprosy (P = 0.0344).

CONCLUSIONS

These findings suggest that Tregs play a relevant role in the etiopathogenesis of leprosy, mainly in type I leprosy reaction.