Development and In Vitro Evaluation of Long Circulating Liposomes for Targeted Delivery of Gemcitabine and Irinotecan in Pancreatic Ductal Adenocarcinoma.

The classically used nontargeted chemotherapeutic approach to pancreatic cancer has a dual drawback of suboptimal drug delivery at the target site and the systemic side effects produced by the unfettered exposure of the drug to healthy tissue. This study has the objective of developing novel poly(2-ethyl-2-oxazoline) (PETOX)-based long circulating liposomes loaded with gemcitabine and irinotecan for the treatment of pancreatic ductal adenocarcinoma, with a juxtaposition to PEGylated and uncoated liposomes. A PETOX-cholesteryl chloroformate lipopolymer conjugate (PETOX-ChC) with a carbonate linkage was prepared and characterized by 1H NMR, FTIR, and DSC. Liposomes were prepared using the thin film hydration technique followed by freeze-thaw and membrane extrusion methods. Liposome characterization includes particle size determination, zeta potential determination using a zetameter, and structural elucidation using 31P NMR and cryo-TEM. The PETOXylated liposomes showed a particle size of 180.1 ± 2.2 nm and a zeta potential of - 33.63 ± 1.23 mV. The liposomal combination therapy of gemcitabine and irinotecan was found to have an IC50 value 39 times lower in comparison to the drug combination in solution, while the PEGylated and PETOXylated liposomes showed IC50 values 1.6 times lower and 2 times lower than that of uncoated liposomes, respectively, against Mia PaCa II pancreatic cancer cell line. The PEGylated and PETOXylated liposomes showed 4.1 and 5.4 times slower macrophagial uptake in vitro in comparison to the uncoated liposomes respectively. The PEGylated liposomes showed 11% higher in vitro macrophagial uptake in comparison to PETOXylated liposomes.

Role of 30 kDa antigen of enteric bacterial pathogens as a possible arthritogenic factor in post-dysenteric reactive arthritis.

BACKGROUND: Reactive arthritis (ReA)/Reiter's syndrome (RS) may be caused as a sequel of infections caused by enteric bacterial pathogens, although the mechanisms through, which different pathogens cause similar disease are not clear. AIM: This study was done to look for the presence and role of any common bacterial antigen among the pathogens isolated from such patients. MATERIALS AND METHODS: A total of 51 patients of ReA and 75 controls (three groups of 25 subjects each: Group 1: Patients who did not develop arthritic complications within 3 months after bacillary dysentery/diarrhea; Group 2: Patients with other arthritic diseases and Group 3: Normal healthy subjects) were included. The isolated enteric pathogens were tested to detect the immunodominant antigens. RESULTS AND CONCLUSIONS: A common 30 kDa antigen was found to be specifically present among seven arthritogenic enteric bacterial strains belonging to three genera, Salmonella, Shigella and Hafnia. Post-dysenteric ReA patients' sera show higher levels of immunoglobulin G, immunoglobulin M and immunoglobulin A antibodies against this antigen as compared to the controls. Lymphocytes of ReA patients recognize this antigen, proliferate and produce interleukin-2 in response to this antigen more than the lymphocytes of controls. 30 kDa antigen may be a common arthritogenic factor associated with post-dysenteric ReA/RS. The association of Hafnia alvei with post-dysenteric ReA is described for the first time. Four cases of mycobacterial ReA had an association with this antigen, suggesting that the arthritogenic antigen of mycobacteria and enteric bacteria may be of a similar nature.

Long term effects of lithium on glomerular filtration rate in Indian subjects - a cross sectional study.

Glomerular filtration rate (GFR) was evaluated in thirty patients on lithium and in thirty healthy prospective kidney donors by single compartment, multiple sample plasma clearance method using (99m)Technetium diethylene triamine pentaacetic acid ((99m)TC-DTPA). Normality test revealed that dose and treatment duration were skewed and the coefficient of skewness were 0.067 (p< 0.0001) and 1.41 (p< 0.0001) respectively. Age was marginally skewed (p =0.04) for the control group. At 5% significance level, dose and creatinine were negatively correlated (r=-0.030), whereas age and duration were positively correlated (r =+ 0.53) (single tailed only). Duration and GFR seems to be negatively correlated (r = -0.23), however this correlation did not reach statistically significance level. In the present cross sectional study no significant difference in mean GFR was observed in lithium treated affective disorder patients when compared with the age matched normal subjects.

Thyroid functions in lithium-treated psychiatric patients: a cross-sectional study.

In the present cross-sectional study, thyroid functions (viz. thyroid radioiodine uptake [RAIU] and serum T3, T4, and thyroid-stimulating hormone [TSH]) were evaluated in 24 healthy controls and 132 outdoor affective disorder patients. Eleven of these patients were to receive lithium (Li) and the remaining 121 patients were at different stages of Li treatment ranging from 0.7 to 240 mo. RAIU was found to increase significantly throughout the Li therapy and was associated with the corresponding rise in TSH levels. In totality, Li treatment induced subclinical hypothyroidism in 51/132 (39%) of patients. However, 8/51 patients who belonged to known iodine-deficient belt had abnormally high TSH (range 15.2-76.0 microIU/mL), low T4 (5.3+/-2.5 microg/dL), and normal T3 and at least 4 of these 8 patients were clinically hypothyroid. T4 levels declined significantly (p < 0.05) with Li treatment ranging from 61 to 240 mo as compared to the corresponding values in the pre-Li group. The T3/T4 ratio was found to be significantly higher with Li treatment ranging from 0.7 to 6 mo in comparison with the pre-Li group and this value returned to base levels after long-term Li therapy. High T3 and T4 were observed in 13% and 12% of the patients, respectively, as compared to the corresponding control values.

Epstein-Barr virus induced non-Hodgkin's lymphoma as a presenting manifestation of acquired immune deficiency syndrome.

A 30 year old, human immunodeficiency virus (HIV) positive patient presented with fever and intra-abdominal lymphadenopathy. Cytology smears from the nodes showed a high grade Non-Hodgkin's lymphoma (NHL) which was B cell in origin. NHL was the acquired immune deficiency syndrome (AIDS) defining disease in this patient. Polymerase chain reaction (PCR) studies on tumour tissue showed presence of Epstein Barr Virus.

Cutaneous vasculitis in a patient with primary Sjogren's syndrome.

Sjogren's syndrome (SS) is an uncommonly described disorder from India. In this report we describe a patient with SS and who developed cutaneous vasculitis.

Neutrophil functions in rheumatoid arthritis.

Neutrophils play an important role in the pathogenesis of rheumatoid arthritis by accumulation and liberation of active proteolytic enzymes. Despite the active participation of the neutrophils, the patients afflicted with rheumatoid arthritis are prone to multiple infections. We studied neutrophil functions in 20 rheumatoid arthritis patients in active disease and equal number in remission and 20 healthy normal controls. No change in neutrophil function was seen in patients in remission. Phagocytic capacity of the neutrophils in active disease was found to be significantly reduced (p < 0.05). This inversly correlated with the rheumatoid factor (r = -0.128, p = 1). Random migration and chemotaxis was statistically reduced when compared with either healthy controls (p < 0.01) or when compared with patients in remission (p < 0.01). The chemotaxis inhibition was further enhanced by autologus serum (p < 0.05). The serum from patients with active disease also reduced chemotaxis of neutrophils from normal individuals (p < 0.01), indicating reduced cellular response as well as inhibitors in serum. The positive correlation (r = 0.466, p < 0.01) with rheumatoid factor, suggests the inhibitory activity may be due to the circulating rheumatoid factor in the active disease. The postulate that prior saturation of neutrophil receptors with immune complexes lower phagocytosis as well as chemotaxis is sustained. Destruction of chemotaxis receptors by release of various strong oxidative enzymes by neutrophils may also be a factor. Normal leucocytes are seen to take up immunoglobulins from diseases serum but not from normal serum. This uptake of diseased serum may be responsible for reducing the chemotactic and phagocytic function of neutrophils and hence increased incidence of infection in these patients.

Elevated urinary nitrite and citrulline levels in patients with rheumatoid arthritis.

The objective of this research was to determine, if NO production, as measured in the serum and urine, is increased in patients with rheumatoid arthritis. Forty-seven patients with RA were recruited in the study and subdivided into inactive and active disease (24 and 23 patients, respectively). Twenty-eight healthy individuals served as controls and nine patients with gastroenteritis were studied to validate the technique of measurement of NO production. Nitrite and citrulline were measured by spectrophotometry, as surrogate markers of NO production. It was found that serum nitrite and citrulline levels of patients with gastroenteritis were not significantly different from controls and the two subgroups of RA. Urine nitrite and citrulline levels were significantly higher in patients with gastroenteritis as compared to the two subgroups of RA and controls (p > 0.001). Serum and urine nitrite levels of patients with active RA were higher than controls and patients with inactive disease (p > 0.05). Serum citrulline levels were not significantly different among the two subgroups of patients with RA. However, they were significantly higher in patients with active disease as compared with controls (p > 0.05). Urinary citrulline levels were significantly higher among patients with active disease as compared to controls and patients with inactive RA (p > 0.05). It is therefore suggested that urinary nitrite and citrulline levels can be useful for the measurement of NO production and are associated with active disease in patients with RA.

Nitric oxide synthesis is increased in patients with systemic lupus erythematosus.

Nitric oxide (NO) is believed to have a role in the inflammatory process. NO production was measured in 26 patients with systemic lupus erythematosus (SLE) and 20 healthy volunteers, using spectrophotometrically determined serum nitrite and citrulline as surrogate markers. Both nitrite and citrulline levels were significantly higher in patients with SLE than in controls (P < 0.001). Twelve and 10 patients, respectively, with SLE had nitrite and citrulline levels that were two standard deviations higher than the mean level of controls. These patients had a significantly higher measure of disease activity (SLE Disease Activity Index). These data show that there is increased NO production in SLE and that it may serve as a marker for disease activity.

Elevated levels of anti-proteus antibodies in patients with active rheumatoid arthritis.

We carried out this study to determine if our patient population with rheumatoid arthritis (RA) has elevated levels of antibodies to gut bacteria. Seventy patients with RA were categorised as being either active or inactive on clinical grounds. Antibodies to the H and O antigens of Proteus mirabilis and Salmonella typhi were determined by tube agglutination method in these patients, 18 patients with osteoarthritis and 82 healthy controls. There was no significant difference in the anti-proteus antibody titres between both the control groups and patients with inactive disease. However, antibody levels among patients with active disease were significantly higher than controls (P < 0.001). There was no significant difference in anti-salmonella antibody titres among the various disease and control groups. Elevated antibody levels could suggest a role for Proteus as an etiological agent in RA.

Lack of antinuclear antibody in children with atopic dermatitis.

Antinuclear antibody (ANA) was assayed in 76 children with atopic dermatitis (AD) of which 46 were males and 30 females. Their ages ranged from 6 months to 12 years (mean 3.4 years). Age at onset of AD ranged from 2 months to 5.5 years (mean 1.9 years) and its duration ranged from 4 months to 4 years (mean 1.2 years). While facial lesions were present in 56 (73.3%) patients, 49 (64.5%) patients had predominant involvement of extensors. As per severity score designed by Rajka and Langerland, 31 (40.8%), 42 (55.3%) and 3 (3.9%) patients had mild, moderate and severe diseases respectively. History of photosensitivity was present in 6 (7.9%) patients. Serum samples were positive for ANA in a very low titre (1:20) in 2/6 patients with facial lesions. However LE cell, rheumatoid factor and C-reactive proteins were negative and serum complement levels were within normal limits.

Immunogenicity of the nonrepetitive regions of the circumsporozoite protein of Plasmodium knowlesi.

The circumsporozoite antigen (CS) of the simian malarial parasite Plasmodium knowlesi consists of tandemly repeated immunodominant peptide units that are variable and may play a role in evading the immune system. To study the immunogenicity of this antigen in the absence of the immunodominant repeats, the entire nonrepetitive region of the antigen was expressed in Escherichia coli as two fusion proteins with glutathione-S-transferase (GST) representing the amino terminal (GST-CSN) and the carboxy terminal domains (GST-CSC) of the CS antigen. The immunogenicity of these fusion proteins was studied in rabbits and different strains of mice. Antibody raised against both the CSN and CSC domains in both rabbits and every strain of mice recognized the native protein, as detected by immunofluorescence assay (IFA) using P. knowlesi sporozoites. A positive IFA reaction was also obtained with P. vivax sporozoites using antisera raised against the CSC domain. High titer antisera were raised in rabbits against both the domains, whereas mice showed comparatively low titers. On Western blots, mice showed specific response against the CSC domain. In both rabbits and mice, significant titers of antibodies were raised against region II, which has been shown to be the putative sporozoite binding site for hepatocytes in the case of P. falciparum.

Plasma reactive nitrogen intermediate levels in patients with clinically active rheumatoid arthritis.

We studied reactive nitrogen intermediate levels in 31 patients with active rheumatoid arthritis (RA) taking indomethacin and 20 healthy controls using nitrite and citrulline levels, measured by spectrophotometry, as markers. Twenty patients with RA were followed up after 4 and 8 wk of treatment with additional therapy in the form of methotrexate. Mean nitrite levels in 31 patients were 0.94 +/- 0.41 mumol/ml and 20 controls it was 1.18 +/- 0.99. After treatment with methotrexate for 4 and 8 wk the levels were 0.9 +/- 0.45 and 1.25 +/- 1.15 mumol/ml, respectively. Mean citrulline levels in all patients was 1.68 +/- 0.11 and controls was 1.39 +/- 0.6 mumol/ml. Following therapy with methotrexate for 4 and 8 wk the levels were 1.40 +/- 0.49 and 1.40 +/- 0.51 mumol/ml, respectively. It is possible that serum levels of these products may not reflect alterations in the synovial fluid levels. Alternatively, whatever lowering may have been achieved by the anti-inflammatory effect of the therapy may have been countered by drug derived free radicals.

Lack of expression of c-myc oncogene in peripheral blood mononuclear cells of patients with active rheumatoid arthritis.

We looked for the expression of c-myc oncogene, one of the genes that enhance apoptosis, in 11 patients with active rheumatoid arthritis (RA) along with five patients with osteoarthritis (OA) knee as disease controls and six healthy volunteers. A dot-blot assay using a probe specific for c-myc oncogene was performed on total RNA obtained from peripheral blood mononuclear cells. There was no expression in patients with active RA and healthy volunteers. One patient with OA expressed c-myc. Lack of expression of c-myc suggests that in active RA circulating lymphocytes are not in the replicative phase despite ongoing disease activity.

Activation of alveolar macrophage TNF and MCP-1 expression in vivo by a synthetic peptide of C-reactive protein.

Administration of multilamellar vesicles (MLV) encapsulating a synthetic peptide (RS-83277) derived from human C-reactive protein (CRP) augments anti-tumor activity of murine alveolar macrophages and reduces established pulmonary metastases of experimental tumors. To explore mechanisms involved in these phenomena, we investigated cytokine and integrin (CDllb) expression of bronchoalveolar lavage (BAL)-derived alveolar macrophages in control (blank MLV) and RS-83277-MLV-treated C57BI mice. Alveolar macrophage production of tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant bioactivity increased at 48 h after treatment with RS-83277-MLV but not control MLV. Chemoattractant activity was neutralized by antibody to monocyte chemoattractant protein-1 (MCP-1), but not irrelevant immunoglobulin G(IgG). Changes were reflected by augmented TNF-alpha and MCP-1 mRNA levels in pulmonary tissue and enhanced CD11b expression on mononuclear leukocytes derived from total lung tissue, but not on BAL-derived alveolar macrophages. Results suggest that RS-83277-MLV treatment is associated with activation of alveolar macrophage TNF-alpha and MCP-1 production and up-regulation of adhesion molecules on pulmonary mononuclear leukocytes but not on alveolar macrophages.