Raman and X-ray photoelectron spectroscopic investigation of solution processed Alq3/ZnO hybrid thin films.

In this study, we characterize the solution processed tris-(8-hydroxyquinoline)aluminum(Alq3)/ZnO hybrid thin films using Raman and X-ray photoelectron spectroscopic (XPS) techniques. Raman studies reveal the sol-gel derived spin-coated ZnO thin film has hexagonal wurtzite structure. The incorporation of Alq3 molecules in the hybrid film results in the formation of bonding onto the surface of highly crystalline ZnO nanoparticles. The XPS confirms the incorporation of Alq3 in the hybrid thin films and corroborates that the Alq3 molecules may be adsorbed onto the surface of ZnO nanoparticles (chemisorption), showing the existence of chemical interaction between Alq3 and ZnO in the hybrid films. These studies support that the chemisorbed Alq3 molecules onto the ZnO nanoparticles may facilitate the charge transfer (non-radiative) between Alq3 and ZnO in the hybrid thin films which will be useful to enhance the optical and electrical properties for the optoelectronic device applications.

Phenotypic characterization of a pair of molecules in tissues confer to classical Mendelian or non Mendelian ratios.

Studies have reported a wide range of inflammatory responses in the nerve, skin and plasma of leprosy patients. The expression levels of each biomolecule was individualistic, however could be categorized as high and low based on their statistical mean level. Here we report for the first time, expression of a set of biomolecules relating with each other in a defined proportion. The hypothesis of this paper is that the segregation of high and low combinations of a set of biomolecules follows either classical Mendelian dihybrid ratio or epistatic ratios. This hypothesis was tested for 17 molecules in three tissues; nerve, skin and plasma and were confirmed to interact in 9:7, 9:3:4, 12:3:1, 13:3, 15:1 epistatic proportions. These findings suggest that there could be a significant role of networking of molecules in defined epistatic proportions and could be important in pathophysiology of peripheral nerve.

The significance of facial patches and type I reaction for the development of facial nerve damage in leprosy. A retrospective study among 1226 paucibacillary leprosy patients.

Charts of 1226 paucibacillary leprosy patients, registered between 1982 and 1987 were reviewed for recent facial nerve damage, facial patches and the presence of Type I reaction. Twenty-six (2.1%) patients with recent lagophthalmos were identified. In a great majority (85%) patients with recent lagophthalmos showed significant patches over the malar region or around the eye, at the same side as the nerve damage together with clinical signs of Type I reaction. This combination of significant patches in certain locations and Type I reaction seems to be a pre-condition for facial nerve damage. The clinical implication is that a small group of patients may be identified, who are at risk of facial nerve damage. By examining these patients more carefully it will be possible to detect nerve damage early and to prevent permanent damage of the facial nerve by timely treatment with an appropriate steroid regimen.

Treatment of recent facial nerve damage with lagophthalmos, using a semistandardized steroid regimen.

Twenty-seven patients with borderline leprosy and facial nerve damage of less than or equal to 6 months duration (36 eyes) were treated with a semistandardized regimen of steroids (the average starting dose was 25-30 mg, duration 5-6 months) on an outpatient basis. Red and raised reactive patches were usually present in the upper malar area or around the eye(s) in patients with recent lagophthalmos. The lid gap was measured in millimetres during gentle and strong closure. After completion of the steroid course 75% of the eyes had complete closure or only a slight gap of less than or equal to 2 mm on gentle closure. Steroids were found to be beneficial and safe, in the dosage that we prescribed.

Clinical and histopathological activity in paucibacillary leprosy patients after fixed-duration multidrug therapy.

In 37 clinically-diagnosed borderline-tuberculoid (BT) leprosy patients skin biopsies were done prior to starting multidrug therapy (MDT) and at the end of 6 months therapy. Clinical and histopathological activity, graded as active, resolving and inactive, were studied at the end of 6 months of MDT. Of the 37 clinically-diagnosed BT patients 24 could be confirmed by histopathology as having BT leprosy, while the other 13 biopsies showed features of indeterminate (I) leprosy. After 6 months of MDT, out of the 24 histopathologically-confirmed BT patients, 4 (17%) showed clinical activity and 8 (33%) showed histopathological activity. Of the 13 histopathologically-diagnosed indeterminate cases all were clinically inactive but histological activity persisted in 3 cases (23%). Out of the 37 clinically-diagnosed BT patients 3 showed both clinical and histopathological activity at the end of MDT. This study emphasizes the importance of performing histopathological examinations on leprosy patients undergoing research studies for the confirmation of diagnosis and for proper classification of the disease. The histopathological activity that outlasts the MDT may be due to the bacillary fragments that persist but clinical activity coupled with histopathological activity seen in 3 patients at the end of 6 months may foreshadow a relapse and these patients and others like them need to be followed up for longer durations.

Histological studies in primary neuritic leprosy: changes in the apparently normal skin.

The visually normal skin of 196 patients diagnosed clinically to have primary neuritic leprosy was studied histologically to determine whether there were any specific changes due to the disease in this site. Histological changes due to leprosy were seen in 32.1% of the patients, and included, indeterminate leprosy in 19.4%, borderline tuberculoid leprosy in 6.6% and borderline lepromatous leprosy in 6.1%. The remaining biopsies showed mild non-specific dermal inflammation, mild nerve changes or no significant lesion. The nerve inflammation and/or granulomas were mostly in the deep dermal nerves or neurovascular complexes. This study shows that there is a cutaneous component to primary neuritic leprosy and the disease is not totally confined to nerves. The absence of visible hypopigmented patches in these patients is probably related to the deep location of the dermal inflammation.

Childhood leprosy in an urban clinic, Hyderabad, India: clinical presentation and the role of household contacts.

A retrospective case note study was done of children below the age of 14 years who attended Dhoolpet Leprosy Research Centre (DLRC) over the decade 1990-1999. The aim of the study was to describe the pattern of clinical presentation, the role of household or near neighbour contacts and the incidence of neuritis and reactions. In all, 3118 leprosy patients were registered during this period, of whom 306 were children [182 (60%) male]; 95 children had a single patch, 159 had five or fewer than five patches and 37 had multiple patches. The youngest case detected was 9 months old. The spectrum of leprosy in these children was: TT 62 (20.3%); BT 203 (66.3%); BB 3 (1%); BL 23 (7.5%); LL 5 (1.6%) and PNL 10 (3.3%). Twenty-nine cases (9.4%) were smear positive. Ninety-one children (29.7%) developed a reaction, 86 type I and five type II. A history of contact was present in 119 (38.8%) cases, family contact in 113 (95%) and other than family in six (5%). Classification of the contact was available in only 60 patients. Among the contacts of the index case, 21 (35%) suffered from PB leprosy and 39 (65%) from MB leprosy. All contacts were from the immediate family. This study shows that childhood leprosy cases continue to present in significant numbers to this outpatient clinic. There is a high level of family contact with leprosy in these cases, strengthening the strategy of screening children in leprosy-affected households. The high incidence of reactions and nerve damage in children emphasizes the importance of early detection and treatment.

Histological studies in primary neuritic leprosy: changes in the nasal mucosa.

The nasal mucosae of 39 cases of primary neuritic leprosy (PNL) registered at Karigiri were studied histologically to determine nasal mucosal involvement in PNL and its relevance to the pathogenesis of the disease. Specific changes of leprosy were seen in 20 (51%) biopsies, ranging from macrophage granulomas with acid fast bacilli, to epithelioid granulomas and nerve inflammation. The remaining biopsies revealed chronic inflammatory changes of the mucosa or mild non-specific nerve changes. These findings show that there are widespread effects of the disease even in PNL patients in whom the disease is believed to be confined to the peripheral nerves. The findings also show that early leprosy involvement can be found in the nasal mucosa even before lesions become apparent in the skin or other parts of the body. The nasal mucosa could be one of the sites for the primary lesion in leprosy. Clinical and histological examination of the nasal mucosa may be useful and important in the early diagnosis of leprosy and especially in contacts.

Detection of superoxide dismutase isozymes in normal and X-irradiated chick amniotic fluid by using isoelectric focussing.

Isoelectric focussing of amniotic fluid of chick system over pH gradient of 4-6 revealed presence of 8 superoxide dismutases. The superoxide dismutase (SOD) isozymes were identified as one major isozyme and other seven were minor ones. Higher expression of SOD (major isozyme) with exposure of 1000 R was studied. The isoelectric point (pI) of major SOD isozyme in control (5) differs from radiation exposed samples (5.12). Hence in radiation exposed amniotic fluid showed more pronounced SOD major isozyme, than control amniotic fluid. It is clearly evident that the SOD isozyme with X-irradiation having different isoelectric points, may lead to modification of the amino acid composition and charges of amino acids in the protein structure of SOD. So, the higher SOD isozyme expression has a role in defence action against free radical damage by X-irradiation during embryonic development.

Bacteriological study of pus isolates from neuropathic plantar ulcers associated with acute inflammatory phase.

In this retrospective study, sensitivity of organisms cultured from ulcers of leprosy patients without and with diabetes mellitus, diabetic patients without leprosy and patients with ulcers from other causes was examined. The profile of organisms grown from these groups of patients did not differ significantly. However, there was a high prevalence of organisms like Proteus, E. coli and Enterococcus in the ulcers of leprosy patients indicating faecal contamination of the ulcers. Co-trimaxazole and tetracycline were of little value in the treatment of these ulcers. We therefore recommend that in situations where there is no culture facility, the patients be started on a course of penicillin and gentamycin. If these antibiotics fail, it would be necessary to use more advanced antibiotics like norfloxacin, amikacin and ciprofloxacin.

The histological diagnosis of leprosy type 1 reactions: identification of key variables and an analysis of the process of histological diagnosis.

BACKGROUND: Type 1 leprosy reactions (T1R) are a major inflammatory complication of leprosy affecting 30% of patients with borderline leprosy, but there has been no diagnostic evaluation of the histological diagnosis of this entity. METHODS: In a prospective study based in India, skin biopsies were taken from 99 patients with clinically diagnosed T1R and 52 non-reactional controls. These were assessed histologically by four histopathologists whose assessments were then compared. RESULTS: Reactions were under-diagnosed, with 32-62% of clinically diagnosed reactions being given a histological diagnosis. The pathologists showed good specificities (range 72% to 93%) but much poorer sensitivities (range 42% to 78%). The most commonly reported histological features of TIR were cell maturity, oedema and giant cells. Five key variables were identified that the pathologists used in diagnosing a reaction: intra-granuloma oedema, giant cell size, giant cell numbers, dermal oedema and HLA-DR expression. A predictive model for the diagnosis of T1R was developed using stepwise logistic regression analysis, with clinical diagnosis of reaction as an outcome, and then identification of the key variables that each pathologist used in making the diagnosis of T1R. 34-53% of the variation between pathologists could be accounted for. The four pathologists used a similar diagnostic model and for all of them their estimations of epithelioid cell granuloma oedema, dermal oedema, plasma cells and granuloma fraction were significant variables in the diagnosis of T1R. Each pathologist then added in variables that were specific to themselves. CONCLUSIONS: This study has identified T1R as being under-diagnosed in comparison with clinical assessments. Key variables for diagnosing T1R were established. This comparative masked study highlights the need for such studies in other inflammatory conditions.

Histological changes in the nerve, skin and nasal mucosa of patients with primary neuritic leprosy.

Primary neuritic leprosy (PNL) presents as a peripheral neuropathy with no visible skin patches and skin smears negative for acid fast bacilli. The pathogenesis of PNL is poorly understood. The aim of the study was to document the histological changes in the nerve, apparently normal skin and nasal mucosa in PNL and to study its significance to the pathogenesis of leprosy lesions. The study is based on a cohort of 208 PNL patients registered at the Schieffelin Leprosy Research and Training Centre, Karigiri. All patients had a nerve biopsy, 196 had a skin biopsy and 39 had a nasal mucosal biopsy. The findings reveal that PNL patients exhibit a spectrum of disease histologically in the nerve ranging from lepromatous to tuberculoid leprosy with a significant proportion (46%) manifesting a multibacillary leprosy histology. Findings in the apparently normal skin and nasal mucosa reveal that there are widespread changes due to leprosy in tissues such as the skin and nasal mucosa even when the disease appears clinically confined to a few nerves. PNL may be an early stage in the pathogenesis of the disease before the appearance of skin lesions. The number of nerves enlarged and lepromin status did not give any clue to the nature of underlying disease.

Multibacillary nerve histology in clinically diagnosed borderline tuberculoid leprosy patients.

The classification of leprosy into multibacillary (MB) and paucibacillary (PB) patients in almost all clinics is entirely dependent on clinical examination. In a study of 21 patients clinically classified as borderline tuberculoid (BT) and, therefore, belonging to the PB group, skin smears and skin and nerve biopsies were examined. Four patients did not have any histopathological evidence of leprosy. Skin smears showed that 1 patient was positive for acid-fast bacilli (AFB), 2 skin biopsies belonged to the borderline lepromatous (BL) category and showed AFB in their lesions, and AFB were present in 10 nerve biopsies classified as BL. It is possible that reported relapses among PB patients may be in those patients with demonstrable AFB in the lesions, including nerves. A careful follow-up study of this particular group of patients after PB multidrug therapy is suggested to resolve this question.

Quantitative assessment of facial sensation in leprosy.

The trigeminal and great auricular nerves which supply sensation to the face are affected in leprosy. No objective sensory testing methods have been devised for testing sensation in the face. Testing for corneal sensation to ascertain trigeminal nerve or visualization and palpation of the great auricular nerve alone may not be enough to establish the involvement of these nerves. In a sample of leprosy patients, face sensation threshold measurements were done using a set of three Semmes-Weinstein (SW) monofilaments that gave a force of 0.05-0.07, 0.2 and 2 g. Sensation was tested by three examiners and intra- and inter-observer testing was used as a means to validate the findings. Within the limitations of this study, the results indicate that use of SW monofilaments is a fairly reliable and repeatable method for sensory testing in the face. During follow up, a single filament with a force of 0.5-0.7 g (2.83 marking number in SW filament or any other filament with a corresponding gram force) could be used to assess sensation. A simple procedure of quantifying sensation in these nerves is suggested. A method to incorporate trigeminal or great auricular nerve sensory testing into the existing sensory assessment charts is also discussed.

Immunohistochemical analysis of cellular infiltrate and gamma interferon, interleukin-12, and inducible nitric oxide synthase expression in leprosy type 1 (reversal) reactions before and during prednisolone treatment.

The effects of prednisolone treatment on the cellularity and cytokine (gamma interferon, interleukin-12, and inducible nitric oxide synthase) profiles of leprosy skin type 1 (reversal) reactions were studied using immunohistochemistry. Skin biopsies were taken from 15 patients with leprosy type 1 (reversal) reactions at days 0, 7, 28, and 180 after the start of steroid treatment. Prednisolone treatment had little effect at day 7, but by day 28 significant decreases were found in cytokine levels. Some patients maintained cytokine production at days 28 and 180. These results illustrate the strong Th1 profile of type 1 reactional lesions, the slow response to steroid therapy, and continuing activity at 180 days.

M. leprae binds to a 28-30-kDa phosphorylated glycoprotein of rat peripheral nerve.

To understand Mycobacterium leprae-peripheral nerve interaction, we have investigated the binding of M. leprae to rat peripheral nerve proteins in an in vitro model using 32P-phosphorylated proteins of the peripheral nerve. Intact M. leprae binds to a major phosphorylated protein of 28-30 kDa and, to a minor extent, to a few proteins of molecular weight 45-55 kDa. This binding was more specific for M. leprae since only insignificant binding was observed with other bacteria, such as M. bovis or Escherichia coli. M. leprae did not show binding to several phosphorylated proteins of the rat brain. The 28-30-kDa binding protein of the rat peripheral nerve was found to be a glycoprotein by concanavalin A-Sepharose column chromatography.

Expression of CC and CXC chemokines and chemokine receptors in human leprosy skin lesions.

We have investigated the expression of chemokines and their receptors in leprosy skin lesions using immunohistochemistry. Skin biopsies from 25 leprosy patients across the leprosy spectrum, 11 patients undergoing type I reversal reactions and four normal donors were immunostained by ABC peroxidase method using antibodies against CC and CXC chemokines and their receptors. Using an in situ hybridization technique we have also studied the expression of monocyte chemoattractant protein 1 (MCP-1), RANTES and interleukin (IL)-8 chemokines mRNA in leprosy skin lesions. Chemokines and receptor expression was detected in all leprosy skin biopsies. Expression of CC chemokines MCP-1 (P < 0.01) and RANTES (P < 0.01) were elevated significantly in borderline tuberculoid leprosy in reversal reaction compared to non-reactional borderline tuberculoid leprosy, but there was no difference in the expression of IL-8 chemokine. Surprisingly, there was no significant difference in the expression of CC (CCR2 and CCR5) and CXC (CXCR2) chemokine receptors across the leprosy spectrum. Similarly, there was no significant difference in the expression of mRNA for MCP-1, regulated upon activation normal T cell expressed and secreted (RANTES) and IL-8 chemokines. Here, the presence of a neutrophil chemoattractant IL-8 in leprosy lesions, which do not contain neutrophils, suggests strongly a role of IL-8 as a monocyte and lymphocyte recruiter in leprosy lesions. These results suggest that the chemokines and their receptors, which are known to chemoattract T lymphocytes and macrophages, are involved in assembling the cellular infiltrate found in lesions across the leprosy spectrum.

Mycobacterium leprae binds to a 25-kDa phosphorylated glycoprotein of human peripheral nerve.

Mycobacterium leprae, the causative agent of leprosy, specifically invades and destroys the peripheral nerve, which results in the main clinical manifestation of the disease. Little is known about the bacteria-nerve protein interaction. We show in the present work that M leprae binds to a 25 kDa glycoprotein from human peripheral nerve. This protein is phosphorylatable and it binds to lectins which have alpha-mannose specificity. This M leprae-protein interaction could be of importance in the pathogenesis of leprosy.