A new clerodane furano diterpene glycoside from Tinospora cordifolia triggers autophagy and apoptosis in HCT-116 colon cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora cordifolia is a miraculous ayurvedic herb used in the treatment of innumerable diseases such as diabetes, gonorrhea, secondary syphilis, anaemia, rheumatoid arthritis, dermatological diseases, cancer, gout, jaundice, asthma, leprosy, in the treatment of bone fractures, liver & intestinal disorders, purifies the blood, gives new life to the whole body; (rejuvenating herb) and many more. Recent studies have revealed the anticancer potential of this plant but not much work has been done on the anticancer chemical constituents actually responsible for its amazing anticancer effects. This prompted us to investigate this plant further for new potent anticancer molecules. AIM OF THE STUDY: The present study was designed to isolate and identify new promising anticancer candidates from the aqueous alcoholic extract of T. cordifolia using bioassay-guided fractionation. MATERIALS AND METHODS: The structures of the isolated compounds were determined on the basis of spectroscopic data interpretation and that of new potent anticancer molecule, TC-2 was confirmed by a single-crystal X-ray crystallographic analysis of its corresponding acetate. The in vitro anti-cancer activity of TC-2 was evaluated by SRB assay and the autophagic activity was investigated by immunofluorescence microscopy. Annexin-V FITC and PI dual staining was applied for the detection of apoptosis. The studies on Mitochondrial Membrane potential and ROS (Reactive oxygen species) production were also done. RESULTS: Bioassay guided fractionation and purification of the aqueous alcoholic stem extract of Tinospora cordifolia led to the isolation of a new clerodane furano diterpene glycoside (TC-2) along with five known compounds i.e. cordifolioside A (ß-D-Glucopyranoside,4-(3-hydroxy-1-propenyl)- 2,6-dimethoxyphenyl 3-O-D-apio-ß-D-furanosyl) (TC-1), ß-Sitosterol(TC-3), 2ß,3ß:15,16-Diepoxy- 4α, 6ß-dihydroxy-13(16),14-clerodadiene-17,12:18,1-diolide (TC-4), ecdysterone(TC-5) and tinosporoside(TC-6). TC-2 emerged as a potential candidate for the treatment of colon cancer. CONCLUSION: The overall study on the bioassay guided isolation of T.cordifolia identified and isolated a new clerodane furano diterpenoid that exhibited anticancer activity via induction of mitochondria mediated apoptosis and autophagy in HCT116 cells. We have reported a promising future candidate for treating colon cancer.

A Traditional NSAID Aspirin along with Clofazimine in Erythema Nodosum Leprosum Reaction: Study of Six Cases.

Six male patients of lepromatous leprosy with erythema nodosum leprosum reaction (ENL) reactions diagnosed clinically and by slit skin smear were treated with aspirin and clofazimine. Aspirin was given in the daily dosage of 75mg/kg body weight up to a maximum of 2.8 grams in four divided doses, along with daily 300mg clofazimine in three divided doses and dapsone 100mg daily with rifampicin 600mg once a month. Aspirin was continued in the same dosage for a month before being tapered and stopped at the end of third month when clofazimine was also reduced to 50 mg daily. All the six cases had an excellent response in 15 days.

Epidemiological trends of leprosy in an urban leprosy centre of Delhi: a retrospective study of 16 years.

This study was done by collecting the retrospective data from 1994 to 2009 of patients attending the urban leprosy centre attached to the department of dermatology, STD & leprosy of PGIMER & Dr. R M L Hospital, New Delhi. The data was analysed according to age, sex, type of leprosy, leprosy reactions, deformities and relapse and compared with the national figures by comparison of proportions after taking the national data per 10,000 population. A total of 3659 patients attended our ULC (Urban Leprosy Centre) among which 2741 were male and 945 females (M:F-3:1). 669 patients (18.2%) were children. The data analysed show a gradual decline in new case detection rate with a marginal rise in 2005 and 2008. Percentage of MB cases was falling consistently till 2005 after which it showed an abrupt rise. The incidence of type 1 reaction varied from 21% in 1994 to 10% in 2009 in PB patients and from 6% in 1994 to 8% in 2009 in MB patients. The trend of type 2 reactions in MB patients showed a slow declining trend. MDT completion rate showed an impressive improvement from 56% in 1994 to 90% in 2009. The number of patients revisiting the ULC with features of relapse also showed a decrease in number. The pattern of visible deformities showed an almost constant trend similar to national figures. Improved MDT completion rate helps in reducing the disease transmission, severity, reactions and disabilities.

Annular vesiculobullous eruptions in type 2 reaction in borderline lepromatous leprosy: a case report.

An untreated case of BL presented with clinical features of type 2 reaction (T2R) confirmed by histopathology. The case was a 18-year-old female with borderline lepromatous leprosy who developed annular vesiculobullous eruptions oversome of the pre-existing plaques on arms and upper back along with fever and severe neuritis after a short course of ofloxacin intake prescribed for urinary tract infection. In addition to the above lesions, some of the existing lesions showed acute exacerbation characterized by erythema, oedema, tenderness and vesiculobullous eruption. This can be considered as an example of leprous exacerbation as described in older literature. T2Rs are common in lepromatous leprosy and not so uncommonly are observed in borderline lepromatous leprosy. The vesiculobullous and crusted lesions developing over the existing borderline plaques, some of them presenting in an annular pattern in T2R in the form of leprous exacerbation, have been reported rarely in the literature.

Inoculation leprosy and HIV co-infection: a rare case with nerve involvement preceding development of skin patch and type 1 reaction as immune reconstitution syndrome following antiretroviral therapy.

The transmission of leprosy has been universally accepted to be primarily, through nasal dissemination from multibacillary patients to the susceptible persons. However, the possibility of leprosy transmission through prolonged skin contact with abraded leprous skin or through skin inoculation can not be ruled out. We report a case of development of a paucibacillary leprosy patch close to the site of a local trauma, after an interval of about 13-14 years, in a HIV positive subject. Also discussed are the various hypotheses in the aetiopathogenesis of leprosy like entry route of lepra bacilli into the body, viability of lepra bacilli in the environment and evolution of skin and nerve lesions of leprosy.

Non-familial multiple keratoacanthomas in a 70 year-old long-term non-progressor HIV-seropositive man.

We describe here multiple keratoacanthomas in an Human Immunodeficiency Virus (HIV)-seropositive 70 year-old man. The patient had multiple epithelial tumors of the skin showing rapid growth, histopathological features of a keratoacanthoma and a conspicuous tendency toward spontaneous remission. A diagnosis of nonfamilial multiple keratoacanthoma was established. The patient had a CD4 count of 633 cells/microL. The HIV disease in our patient was of a nonprogressive nature with CCR5-positive T cells.

Systemic sarcoidosis in a case of lepromatous leprosy: a case report.

The clinical features of cutaneous sarcoidosis and leprosy are some times difficult to differentiate and there have been many reports where pulmonary sarcoidosis was treated as pulmonary tuberculosis or a case of sarcoidosis was treated with anti-leprosy multidrug therapy, before a correct diagnosis was made. So far there has been only one published case report of leprosy and sarcoidosis co-infection, where tuberculoid leprosy developed in a case of sarcoidosis, known for over a decade. We are reporting a case of dual affliction, where sarcoidosis was discovered (on routine screening) in a case of lepromatous leprosy, after administration of 2 years of multidrug therapy. The role of mycobacterial antigens (among a vast array of different animate or inanimate particles) in causation of sarcoidosis, is still speculative, as reviewed from literature.

New lesions after MDT in PB and MB leprosy: a report of 28 cases.

Appearance of new skin and/or nerve lesions during or after fixed duration of multi drug therapy (MDT), both in multibacillary (MB) and paucibacillary (PB) leprosy, is not uncommon. It could be a lesion due to reaction (type 1 or type 2), relapse due to multiplication of persisting or drug resistant bacilli or reinfection due to re-entry of lepra bacilli from outside. It is relatively easier to recognize the lesions due to classical reaction, both clinically and histopathologically. However, the differentiation could be difficult in other situations, especially when many of the relapse cases may present with features of reaction at the onset. Similarly, sometimes in late reversal reaction in addition to development of classical acute inflammation of old lesions, many of the patients developed multiple fresh new lesions without any sign of inflammation. We report a study of group of 28 relapsed leprosy cases, who developed new skin and/or nerve lesions at greatly varying time intervals (3 months to 22 years) after stopping MDT. Of these 28 patients, 11 were MB (1 LL,6 BL and 4 BB) and 17 were PB (12 BT, 4 TT and 1 Neuritic) at their first treatment. They reported to the Urban Leprosy Center (ULC) of Dr R M L Hospital during the period of 5 years (2002-2007). All patients came through self referral, 13 of them (46.4%) had received MDT outside our hospital (regular in 11 cases and irregular in 2 cases, as per the patient's statement), while the rest 15 had received full MDT regularly from our center (irregular in 1 case). All previously 11 MB cases developed new skin lesions of MB type (1 LL to LL, 3 BL to LL, 3 BL to BL, 1 BB to BL and 3 BB to BB). Of the 17 cases PB at their first treatment, 16 developed new lesions of PB type. Out of 4 TT cases, 1 had new lesions of TT, 1 BT and 2 LRR type lesions. Of the 12 BT cases at first presentation, 9 had BT, 1 secondary neuritic and 1 presented as LRR, while 1 BT case had new lesions of BL type. The one pure neuritic leprosy case presented as neuritic case only, after an interval of over 20 years. The post-MDT intervals of appearance of new lesions were 3-6 months in 5 cases (Group A), 8-30 months in 13 cases (Group B), from 3-10 years in 4 cases (Group C) and 15-22 years in 6 cases (Group D). All patients were successfully treated with a second course of MDT, as per the spectrum of the disease according to the number of fresh lesions. The likely cause of new lesions in group A (<6 months interval) could be either (1) mild type 1 reaction or (2) early relapse due to inadequate MDT. Similarly, the new lesions appearing in group B (0.5-3 years) could also represent mild type 1 reaction following improvement of CMI or a true early relapse. The possible causes of early relapse may be because of original misclassification or inadequate chemotherapy/irregular treatment or insufficient duration of therapy. In group C (3-10 years), the cause would most probably be late relapse, the possible causes of late relapse is either due to drug resistance and M. leprae persisters. When the time interval goes beyond 10 years (Group D), as in 6 of our cases, the possibility of reinfection can not be excluded besides causes of late relapse, since this period is usually considered equivalent to the maximum incubation period of lepra bacilli. In lepromatous leprosy, where the specific CMI against M. leprae is highly compromised, there is always a possibility of reinfection as long as the source of infection persists in the community and in such cases immunotherapy would be highly beneficial for prevention of reinfection. The post MDT time interval, lepromin test or drug resistance study both in vitro and in vivo may provide some clue to the mechanism responsible. All doubtful cases of new lesions with clinical presentation of type 1 reaction were diagnosed as relapse, through the therapeutic trial with oral prednisolone for 4-6 weeks and other cases. All cases with new lesions were treated with a second course of MDT (MB or PB) as per classification of new lesions.

Predominance of Mycobacterium fortuitum-chelonae complex in Ghatampur field area, endemic for leprosy.

Non-tuberculous mycobacteria (NTM) are commonly found in the environment. As exposure to environmental mycobacteria has been reported to immunomodulatory in this study, the presence of environmental mycobacteria was investigated in soil, drinking water and drainage sample in Ghatampur, India, which is known for high endemicity for leprosy. Soil, drinking water from the hand pumps/wells and also drainage water collected in pools was collected in clean containers and cultured for environmental mycobacteria. Samples were processed according to the protocol established earlier. 69 soil, 62 drinking water and 31 drainage water samples were analysed from soil and water collected from 48 villages of this field area. After decontamination, cultures were set upon Lowenstein Jensen (LJ) medium. Mycobacteria were identified using biochemical tests and molecular techniques such as PCR-RFLP targeting hsp65 kD and rpoB region as well as 16S ribosomal sequencing in case of isolates showing variable biochemical features. NTM (non-tubercular mycobacteria) were isolated from 47.82% of soil samples, 20.69% of drinking water samples and 19.35% of the drainage water samples, overall mycobacteria could be isolated 52/162 of samples (32.09%). Among these mycobacteria, M. fortuitum-chelonae complex was predominant in this area; other species isolated were M. phlei, M. vaccae, M. terrae and M. flavescens. Relevance of exposure to these mycobacteria on endemicity needs to be studied by immunological and epidemiological parameters.

New lesions after MDT in PB and MB leprosy: a report of 28 cases

Appearance of new skin and/or nerve lesions during or after fixed duration of multi drug therapy (MDT), both in multibacillary (MB) and paucibacillary (PB) leprosy, is not uncommon. It could be a lesion due to reaction (type 1 or type 2), relapse due to multiplication of persisting or drug resistant bacilli or reinfection due to re-entry of lepra bacilli from outside. It is relatively easier to recognize the lesions due to classical reaction, both clinically and histopathologically. However, the differentiation could be difficult in other situations, especially when many of the relapse cases may present with features of reaction at the onset. Similarly, sometimes in late reversal reaction in addition to development of classical acute inflammation of old lesions, many of the patients developed multiple fresh new lesions without any sign of inflammation. We report a study of group of 28 relapsed leprosy cases, who developed new skin and/or nerve lesions at greatly varying time intervals (3 months to 22 years) after stopping MDT. Of these 28 patients, 11 were MB (1 LL,6 BL and 4 BB) and 17 were PB (12 BT, 4 TT and 1 Neuritic) at their first treatment. They reported to the Urban Leprosy Center (ULC) of Dr R M L Hospital during the period of 5 years (2002-2007). All patients came through self referral, 13 of them (46.4%) had received MDT outside our hospital (regular in 11 cases and irregular in 2 cases, as per the patient's statement), while the rest 15 had received full MDT regularly from our center (irregular in 1 case). All previously 11 MB cases developed new skin lesions of MB type (1 LL to LL, 3 BL to LL, 3 BL to BL, 1 BB to BL and 3 BB to BB). Of the 17 cases PB at their first treatment, 16 developed new lesions of PB type. Out of 4 TT cases, 1 had new lesions of TT, 1 BT and 2 LRR type lesions. Of the 12 BT cases at first presentation, 9 had BT, 1 secondary neuritic and 1 presented as LRR, while 1 BT case had new lesions of BL type. The one pure neuritic leprosy case presented as neuritic case only, after an interval of over 20 years. The post-MDT intervals of appearance of new lesions were 3-6 months in 5 cases (Group A), 8-30 months in 13 cases (Group B), from 3-10 years in 4 cases (Group C) and 15-22 years in 6 cases (Group D). All patients were successfully treated with a second course of MDT, as per the spectrum of the disease according to the number of fresh lesions. The likely cause of new lesions in group A (<6 months interval) could be either (1) mild type 1 reaction or (2) early relapse due to inadequate MDT. Similarly, the new lesions appearing in group B (0.5-3 years) could also represent mild type 1 reaction following improvement of CMI or a true early relapse. The possible causes of early relapse may be because of original misclassification or inadequate chemotherapy/irregular treatment or insufficient duration of therapy. In group C (3-10 years), the cause would most probably be late relapse, the possible causes of late relapse is either due to drug resistance and M. leprae persisters. When the time interval goes beyond 10 years (Group D), as in 6 of our cases, the possibility of reinfection can not be excluded besides causes of late relapse, since this period is usually considered equivalent to the maximum incubation period of lepra bacilli. In lepromatous leprosy, where the specific CMI against M. leprae is highly compromised, there is always a possibility of reinfection as long as the source of infection persists in the community and in such cases immunotherapy would be highly beneficial for prevention of reinfection. The post MDT time interval, lepromin test or drug resistance study both in vitro and in vivo may provide some clue to the mechanism responsible. All doubtful cases of new lesions with clinical presentation of type 1 reaction were diagnosed as relapse, through the therapeutic trial with oral prednisolone for 4-6 weeks and other cases. All cases with new lesions were treated with a second course of MDT (MB or PB) as per classification of new lesions.

Does concomitant hiv infection has any epidemiological, clinical, immunopathological and therapeutic relevance in leprosy?

Co-infection with HIV-1 and M. Leprae is a rare event in endemic areas for leprosy and HIV, such as India. Neither an increased HIV prevalence among leprosy cases, nor any rapid progression to AIDS was observed among dual HIV-leprosy infections. The current situation concerning continued new leprosy case-detection and gradual increase in HIV infection in India and a few other developing countries, such as Brazil, emphasizes the importance of monitoring the occurrence of co-infections. There is so far no change in the clinical spectrum of leprosy, PB/MB ratio, leprosy reactions and neuritis among co-infected patients. All types of leprosy occur in HIV patients [except in one study (Borgdorff et al, 1993) where more MB leprosy cases with HIV infection were seen]. Histopathological observations reveal a normal spectrum of appearance in biopsies of leprosy lesions from co-infected patients suggesting that cell-mediated immune response to M leprae is preserved at the site of the disease, despite evidence that these responses are abrogated systemically. All dual infection cases respond to regular treatment, except in three studies which noted more relapses. Therefore, a longer duration of surveillance is advisable after fixed duration therapy, for the detection of early relapse. Type 2 reaction can be managed with a higher dose of clofazimine. Type 1 reaction when developed as such, or as IRIS, needs oral steroids in adequate doses, particularly when associated with neuritis and motor loss, since lower doses may not be able to reverse the motor loss even of early onset. However, higher doses of corticosteroid when given need to be monitored closely. The impact of immune restoration in co-infected patients receiving ART is commonly observed in cases with borderline leprosy.

Penicillamine induced pseudoxanthoma elasticum with elastosis perforans serpiginosa.

Long term D-penicillamine therapy, especially when used to treat Wilson's disease has been shown to cause elastosis perforans serpiginosa, pseudoxanthoma elasticum perforans and other degenerative dermatoses. We report a 23-year-old male patient who presented with multiple firm papules, nodules over the neck, axillae, front of elbows for five years. He was a known case of Wilson's disease on long-term treatment with penicillamine for the past 12 years. The papulonodular lesions were non-tender and some were discrete while others were arranged in a circinate pattern. There was central scarring of the skin within the circinate lesions. In addition, there were several small yellowish papules on both sides of the neck which eventually became confluent to form plaques. Histopathology confirmed the diagnosis of elastosis perforans serpiginosa and pseudoxanthoma elasticum. He was treated with cryotherapy (using liquid nitrogen through cryojet) for former lesions. The lesions showed remarkable improvement after five sittings. Now the patient is under trientine hydrochloride (750 mg twice daily) for Wilson's disease.

Comparative immunological and histopathological study in fifty cases of mucosal/nonmucosal lichen planus.

Lichen planus is a common disorder and 40-50% of LP patients also reveal mucosal lesions. It is well known that mucosal LP lesions take very long to heal in comparison to cutaneous lesions. Rarely erosive mucosal LP can turn malignant. Both CMI and humoral immunity may play role in aetiopathogenesis of LP. Present study was conducted to study and compare CMI, Humoral Immunity, histopathology in mucosal and nonmucosal LP.

Histoid leprosy with ENL reaction.

A 23-year old man presented with firm cutaneous and subcutaneous nodules of histoid leprosy. Some of the nodules suppurated after multidrug therapy (MDT) and these nodules showed features of erythema nodosum leprosum (ENL) on histopathological examination. ENL is a rare phenomenon observed in histoid leprosy.

Subungual malignant melanoma.

A rare case of subungual malignant melanoma in a 43-year-old male, with black dystrophic left middle finger nail plate with positive Hutchison sign is presented. Patient underwent disarticulation at the proximal interphalangeal joint. Histopathology confirmed malignant melanoma, and resection free of tumour cells.

A minisatellite sequence in the upstream region of the DURA3 gene from the halotolerant yeast Debaryomyces hansenii.

The URA3 gene of Debaryomyces hansenii, encoding orotidine 5'-phosphate decarboxylase enzyme, was isolated by complementation in the yeast Saccharomyces cerevisiae. The deduced amino acid sequence is highly similar to Ura3 proteins from other yeast and fungal species. Analysis of the region upstream of the coding sequence revealed the presence of AG-rich minisatellite DNA sequences. In addition, upstream of the DURA3 sequence, we have found the 3'-terminal of a gene encoding a GEA2-like protein.

A PBS2 homologue from Debaryomyces hansenii shows a differential effect on calcofluor and polymyxin B sensitivity in Saccharomyces cerevisiae.

The PBS2 gene encodes a MAP kinase kinase that plays a pivotal role in osmosensing signal-transduction pathway in the yeast Saccharomyces cerevisiae. Mutation in the PBS2 gene has a pleotropic effect. Besides being osmosensitive, pbs2 mutants show altered sensitivity to polymyxin B and calcofluor. Recent studies revealed that Pbs2p plays a different role in osmoadaptation and calcofluor sensitivity. We have isolated a gene homologous to PBS2 from the highly salt-tolerant yeast Debaryomyces hansenii by phenotypic complementation. DNA sequencing of the clone revealed that the gene encoded a protein of 683 amino acid residues. Like Pbs2p, this protein also has a proline-rich motif. Further characterization revealed that this gene could complement polymyxin B sensitivity but did not affect calcofluor sensitivity. Thus, it appeared that Pbs2p also has an independent role in these two physiological processes. The GenBank Accession No. of this sequence is AF371315.

Abdominal hernia following abdominal herpes zoster.

We report a case of abdominal hernia in TIO and 11 segments following herpes zoster at T11 segment.