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The relationship of HLA-DR antigens in rheumatoid arthritis (RA) patients with the presence of rheumatoid factor (RF), extra-articular features, joint erosions, functional status and response to disease modifying anti-rheumatic drugs (DMARDs) was studied. Seventy-four patients with RA were serologjcally typed for class II antigens. Clinical features were recorded before starting DMARDs and during follow-up to assess response and side-effects. HLA-DR1 and DR4 antigens were present more frequently in patients with RA than in the general population. The presence of DR4 correlated with RF seropositivity (P < 0.001) while DR1 was associated with seronegativity (P < 0.02). Ten of the 11 patients with extra-articular features had DR1 and/or DR4. Patients with both DR4 and DR1 had a significantly higher frequency of extra-articular features compared with those with only one of these {P < 0.01). HLA-DR4 and DR1 had no relation with the presence of subcutaneous nodules, erosive changes, deformities, response to drugs and side-effects of DMARDs. [ABSTRACT FROM PUBLISHER]

The aim of the present study was to evaluate two vesicular carrier systems, ethosomes and elastic liposomes loaded with hepatitis B surface antigen, for in vitro qualitative and quantitative uptake by human dendritic cells (DCs) and ability to stimulate T lymphocytes. Quantitative uptake of antigen-loaded carriers was documented by flow cytometry, and internalization of the systems by the DCs was studied using spectral bioimaging. Ability of antigen-pulsed DCs to stimulate autologous peripheral blood lymphocytes and levels of TH1/TH2 cytokines were also examined using flow cytometry. Both vesicular carrier systems as antigen delivery modules and DCs as antigen-presenting cells were able to generate a protective immune response. However, ethosomes were found to have higher internalizing ability and immunogenicity in comparison with elastic liposomes. These properties of ethosomes coupled with their skin-navigating potential, make it an attractive vehicle for development of a transcutaneous vaccine against hepatitis B in preference to elastic liposomes., From the Clinical Editor: Two carrier systems for more potent vaccine administration - ethosomes and elastic liposomes loaded with hepatitis B surface antigen - are compared. Ethosomes demonstrated higher internalizing ability and immunogenicity. Due to their known skin-navigating potential, ethosomes may represent an attractive vehicle for development of a transcutaneous vaccine against hepatitis B., (Copyright 2010 Elsevier Inc. All rights reserved.)

Bhopal gas tragedy is considered as one of the world's worst industrial disaster. Approximately, 3,000-6,000 people died and 200,000 injured due to the leak of 40 tons of methyl isocyanate (MIC) gas from a pesticide plant. We aimed to decipher any persistent and subtle immunotoxic effects of MIC in the survivors of the tragedy. The study was divided into 3 groups i.e. group I (n=40); Age and gender matched non-exposed healthy controls recruited from places within the geographical region of Bhopal but from unaffected zones, group II (n=40); Age and gender matched non-exposed healthy controls recruited from places well outside geographical region of Bhopal and group III (n=40); Age and gender matched MIC exposed subjects from affected zones inside geographical region of Bhopal and the status of inflammatory biomarkers (IL-8, IL-1beta, IL-6, TNF, IL-10, IL-12p70 cytokines and C-reactive protein) were analysed. The results displayed a significant increase in the levels of all circulating inflammatory biomarkers in the MIC exposed group in comparison to non-exposed cohorts. A toxin induced genetic and/or epigenetic alteration seems to be the likely underlying cause. However, further studies are essential for both mechanistic understanding and clinical implications of these patterns.

We have evaluated the efficiency of novel modified liposomes (ethosomes) for transcutaneous immunization (TCI) against Hepatitis B. Antigen-loaded ethosomes were prepared and characterized for shape, lamellarity, fluidity, size distribution, and entrapment efficiency. Spectral bio-imaging and flow cytometric studies showed efficient uptake of Hepatitis B surface antigen (HBsAg)-loaded ethosomes by murine dendritic cells (DCs) in vitro, reaching a peak by 180 min. Transcutaneous delivery potential of the antigen-loaded system using human cadaver skin demonstrated a much higher skin permeation of the antigen in comparison to conventional liposomes and soluble antigen preparation. Topically applied HBsAg-loaded ethosomes in experimental mice showed a robust systemic and mucosal humoral immune response compared to intramuscularly administered alum-adsorbed HBsAg suspension, topically applied plain HBsAg solution and hydroethanolic (25%) HBsAg solution. The ability of the antigen-pulsed DCs to stimulate autologous peripheral blood lymphocytes was demonstrated by BrdU assay and a predominantly TH1 type of immune response was observed by multiplex cytometric bead array analysis. HBsAg-loaded ethosomes are able to generate a protective immune response and their ability to traverse and target the immunological milieu of the skin may find a potential application in the development of a transcutaneous vaccine against Hepatitis B virus (HBV).

We present two cases of chronic idiopathic thrombocytopenic purpura (ITP) on prolonged steroid therapy who developed subcutaneous and brain abscesses due to Nocardia asteroides. The special diagnostic and therapeutic challenges encountered in the patients because of severe thrombocytopenia are being highlighted.

Objective: Primary Sjogren's syndrome (SS) is rarely reported from India. We have studied the clinical spectrum and immunological profile of patients with primary SS., Methods: A prospective analysis of patients with primary Sjogren's syndrome fulfilling San Francisco criteria, seen at our clinic in the last 10 years was carried out., Results: The study included 26 patients, 21 being women. The presenting symptoms included dry eyes, dry mouth, and arthritis/arthralgia. Extra-glandular manifestations were glomerulonephritis, vasculitis, renal tubular acidosis and peripheral neuropathy. The important laboratory abnormalities were hypergammaglobulinaemia (16/20), antinuclear antibodies (18/26), anti-La (11/19) and anti-Ro (10/19). Minor salivary gland provided a definitive diagnosis in 16/26 (60%)., Conclusion: The prevalence of primary Sjogren's syndrome is rare even in tertiary care rheumatology clinics. The clinical and immunological profile as seen here is similar to that reported in Western countries.

Background & Objectives: Although the outcome of children with acute lymphoblastic leukaemia (ALL) has improved dramatically over the last decade, some children still fare poorly and relapses are seen. The sensitivity of leukaemic cells to corticosteroids has emerged as an important prognostic factor in ALL. The t(9,22) translocation, resulting in the bcr-abl fusion gene, is a non-random translocation found in B-lineage acute lymphoblastic leukaemia. It is also known to be an independent poor prognostic factor for long-term disease free survival. We studied the association between the presence of bcr-abl fusion gene and in vitro prednisolone resistance in children with B-lineage acute lymphoblastic leukaemia at diagnosis., Methods: A total of 23 children (aged 1-16 yr, median age: 12 yr) with B-lineage acute lymphoblastic leukaemia at diagnosis were included in the study. The presence of bcr-abl fusion gene was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and the in-vitro resistance to prednisolone was measured by short term colorimetric methyl thiazol tetrazolium (MTT) assay., Results: A median LD50 (lethal dose for 50% cells) for prednisolone in bcr-abl positive children (n=7) was 1.6 mg/ml (range: 0.25-5.0 mg/ml) and that of bcr-abl negative children (n=16) was 0.35 mg/ml (range 0.62-1.0 mg/ml). The median LD50 for prednisolone differed significantly between the bcr-abl positive and negative groups of children with acute lymphoblastic leukaemia (P<0.005)., Interpretation & Conclusion: This is probably the first report to show that leukaemic blasts of bcr-abl positive children with ALL are about four-fold resistant to prednisolone as compared to blasts from bcr-abl negative children. This suggests that one of the reasons for the poor prognosis of bcr-abl positive ALL could be a lower steroid sensitivity.

In vitro resistance to anthracyclines is thought to be a poor prognosis in achieving long-term remission in patients with acute lymphoblastic leukemia (ALL). Expression of a multidrug resistance gene (mdr1) that codes for 170 Kd transmembrane glycoprotein is responsible for conferring resistance to malignant cells to anthracyclines. The t(9:22) translocation, resulting in bcr-abl fusion gene, is commonly found in B-lineage ALL and is known to be a poor prognostic factor for long-term remission. To investigate whether resistance to anthracyclines contributes to poor prognosis in bcr-abl-positive ALL, we studied daunorubicin sensitivity by an in vitro colorimetric methyl tetrazolium (MTT) assay in B-lineage ALL patients who were bcr-abl-positive and compared them with the B-lineage, age-matched bcr-abl-negative group. We also looked for and compared the presence of mdr1 gene expression in these two groups of patients by RT-PCR. Of the 46 patients included in the study, 16 (34.7%) were positive for the bcr-abl fusion gene. mdr1 gene expression was seen in 14 of these 46 patients (30.4%). However, the expression of the mdr1 gene was relatively lower in the bcr-abl-positive group (3 out of 16, 18.7%) compared to the bcr-abl-negative group (11 out of 30, 36.6%). The median LD(50) of daunorubicin (concentration lethal to 50% of the leukemic blasts) differed significantly between bcr-abl-positive and -negative patients (P = 0.018). This in vitro study suggests that bcr-abl-positive ALL is relatively resistant to daunorubicin, but this resistance is not mediated through mdr1 gene expression., (Copyright 2002 Wiley-Liss, Inc.)

Background & Objectives: Drug sensitivity assays are useful in oncology practice for evaluating the sensitivity of malignant cells to anti-cancer drugs. The usefulness of such assays for the prediction of clinical response to therapy has also been demonstrated. The existing methods used for this purpose are time consuming and labour intensive. Here we report a simplified flow cytometry based assay for evaluating the in vitro drug sensitivity of leukaemic cells., Methods: The chemo-sensitivity of three human leukaemic cell lines (a lymphoblastoid cell line, Jurkat; an erythroleukaemic cell line, K 562 and a myelomonocytic cell line HL-60) was investigated by flow cytometry. Flow cytometry was used to determine LD50 (50% inhibitory concentration) for prednisolone on Jurkat and daunorubicin on HL 60 and K 562 cell lines respectively. Per cent cell death could directly be assessed on a flow cytometer by measuring the fluorescence after staining with propidium iodide (PI). For comparison MTT assay was also performed using prednisolone on Jurkat and daunorubicin on HL-60., Results: Cytotoxic effect of drugs was found to be dose dependent. Mean LD50 of prednisolone for Jurkat cells by flow cytometry was 0.805 +/- 0.058 mg/ml and by MTT assay 0.866 +/- 0.115 mg/ml. Mean LD50 of daunorubicin for HL-60 was 1.96 +/- 0.05 micrograms/ml by flow cytometry and 1.90 +/- 0.282 micrograms/ml by MTT assay. The mean LD50 of daunorubicin to K 562 was 0.49 +/- 0.049 mg/ml by the flow cytometry method. The inter-assay variation for the LD50 by flow cytometry based assay was found to be 6, 14 and 10 per cent for Jurkat, HL-60 and K 562 respectively., Interpretation & Conclusion: We report a flow cytometry based drug-sensitivity assay for leukaemic cells, which uses a single dye staining and is rapid, technically simple and reproducible. The results compare well with the more commonly used MTT assay, which is labour intensive and time consuming. The limitation of our method is that it can only be used for studying cells in suspension and is therefore not suitable for adherent cell lines.

The poorer outcome of Indian patients with acute lymphoid leukemia (ALL) has been observed in earlier studies. However, little data is available on their immunophenotypic characteristics. The aim of the present study was thus to characterize the immunophenotypic subsets of Indian ALL patients and correlate with outcome at the end of induction chemotherapy. Immunophenotyping of 45 childhood and 25 adult ALL patients was performed by dual colour flow cytometry using a panel of B-lineage, T-lineage and Myeloid lineage specific monoclonal antibodies. Eighty and 17% of childhood and 92% and none of adults were B-lineage and T-lineage ALL, respectively. Aberrant myeloid marker expression was seen in 11% and 28% of childhood and adult groups, respectively. B-lineage ALL with aberrant T-lineage marker expression was observed in 4.4% and 8% of childhood and adult groups, respectively. Two each induction failures were observed in both childhood and adult groups. All of these were associated with aberrant expression of myeloid and/or T-lineage markers on B-lineage ALL. Aberrant expression of markers was associated with poorer outcome to induction chemotherapy in both childhood and adult ALL patients.

Till date only three series of immunoproliferative small intestinal disease (IPSID) describing 22 patients have been reported from India. Seven patients with IPSID in two tertiary referral centers in India are included in the study. Diagnosis was based on typical clinical features [diarrhoea (7/7), weight loss (7/7), clubbing (6/7), fever (3/7), abdominal pain and lump (3/7)], biochemical evidence of malabsorption and duodenal biopsy findings. All patients were young males (mean age 29.8 +/- 11.8 years, range 17-53). Atypical features included gastric involvement (1/7), colonic involvement (1/7) and appearance of pigmented nails following anti-cancer chemotherapy (1/7) which disappeared six months after omitting doxorubin from chemotherapy regimen. Parasitic infestation was common. Ascaris lumbricoides (1/7), Giardia lamblia and hookworm (1/7), Strongyloides stercoralis and Trichuris trichura (1/7). In the latter patient S. stercoralis became disseminated after anti-malignant chemotherapy. One patient had gastric H. pylori infection. Four of the seven patients who were misdiagnosed as tropical sprue were treated with tetracycline. This raises doubt on efficacy of tetracycline alone in treatment of IPSID. One other patient was misdiagnosed and treated as intestinal tuberculosis. Early diagnosis and administration of chemotherapy may improve survival in this disease.

The monoclonal immunoglobulin (Ig) (M-component) secreted by the tumour plasma cells in multiple myeloma (MM) has specific antigenic determinants (idiotypes; id) that can serve as tumour-specific antigens. The intact Ig molecule is a weak antigen, and small fragments of id protein might be more immunogenic for the induction of id-specific immunity. Dendritic cells (DC) have attracted attention as the most efficient antigen-presenting cells and promising adjuvants for immunotherapy in tumours. In this study the in vitro T-cell response against F(ab')2 and Fab fragments, heavy and light chains of the M-component was examined in five patients with MM clinical stage I. All fragments were able to stimulate T cells but F(ab')2 or Fab fragments and heavy chains induced a stronger response than light chains. DC induced a significantly stronger id-specific immune response than monocytes. Moreover, with DC as antigen-presenting cells, a predominant interferon (IFN)-gamma (type-1 T-cell) response was seen in all patients. Both IFN-gamma and interleukin (IL)-4 (type-1 and type-2 T-cell) responses were noted when monocytes were used. Our study suggests that DC pulsed with idiotypic fragments such as F(ab')2 fragment and heavy chain can be used for the induction of type-1 anti-idiotypic T-cell response for immunotherapy in MM.

Myeloma plasma cells constitute 10% to 90% of the total bone marrow cell count in patients with multiple myeloma (MM). These cells express a variety of cell surface markers, such as HLA-ABC and HLA-DR, and surface antigens that are necessary for professional antigen-presenting cells, including adhesion and costimulatory molecules. In this study, we examined the expression of major histocompatability complex (MHC) and costimulatory molecules on CD38(bright,++) plasma cells in bone marrow aspirates from eight MM patients. Small percentages of plasma cells expressed weak but detectable levels of HLA-DR, HLA-DQ, CD40, CD80, and CD86, which could be upregulated by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha. CD38++ plasma cell and CD38(dim,+) cells were sorted from freshly isolated bone marrow mononuclear cells and tested for their capacity to act as antigen-presenting cells. Indeed, both CD38++ plasma cells and CD38+ cells were able to stimulate allogeneic T cells and present the soluble antigens purified protein derivative and tetanus toxoid to autologous T cells. Recognition of the antigens led to T-cell proliferation and secretion of IFN-gamma and was MHC class-I and -II restricted. Antigen processing and presentation by CD38++ and CD38+ cells were abolished by treatment of the cells with chloroquine. Hence, our study provides for the first time evidence that myeloma plasma cells may act as antigen-presenting cells. Further studies are warranted to examine in detail the molecules required for inducing T-cell stimulation.

Renal disease as the sole manifestation of extra-glandular involvement is rare in primary Sjögren's syndrome. We report a case of membranous nephritis presenting with proliferative changes including crescents, which led to renal insufficiency in a patient with primary Sjögren's syndrome. The renal involvement was not associated with cryoglobulinemia.