Novel approaches to modulate apoptosis resistance: basic and clinical implications in the treatment of chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is an archetype of malignancy resulting from defects in apoptosis. CLL is an exclusive accumulative disorder marked by low proliferative activity and gradual accumulation of clonal B-lymphocytes blocked in the early (G0, G1) phases of the cell cycle. The heterogeneous clinical course indicates diverse in vivo biology of the leukemic cell and suggests that CLL represents diverse behavior. Understanding the molecular biology of the disease has provided insight into the mechanisms that promote tumorigenesis, specifically defective apoptotic signaling pathways. In this review we attempt to provide a comprehensive discussion of CLL including the origin of malignant lymphocytes, the apoptotic defects and the mechanisms leading to disease progression. We further discuss the therapeutic options, focusing mainly on targeted therapy using novel agents. Finally, we suggest future directions for treatment that utilize the understanding of the disease biology.     

Human leukocyte antigen (HLA) class II association with rheumatic heart disease in Pakistan

BACKGROUND AND AIM of the study: Rheumatic heart disease (RHD) is widespread in Pakistan. Specific alleles of the human leukocyte antigen (HLA) system are associated with RHD in various world populations. The study aim was to investigate the involvement of HLA class II alleles in genetic susceptibility to RHD in patients with relatively homogeneous clinical manifestations, in Pakistan. METHODS: Blood samples were collected from 114 unrelated patients (94 females, 20 males) with rheumatic mitral valve disease, predominantly mitral stenosis, as assessed by echocardiography. The control group comprised 109 unrelated, ethnically matched, healthy individuals (60 females, 49 males) with normal echocardiograms. Genomic DNA was extracted from venous blood using a standard phenol/chloroform extraction procedure. HLA-DRB, -DQA1, and -DQB1 alleles were typed using polymerase chain reaction with sequence-specific primers. HLA allele and haplotypes frequencies were then calculated. RESULTS: A significantly higher frequency of DRB1*07 was observed in patients as compared to controls (one-way parametric analysis of variance, F = 4.84, p = 0.028; OR = 1.76, p = 0.039). No alleles for the HLA-DQA1 or -DQB1 loci were associated with the disease. HLA-DRB1*07-DQA1*0501-DQB1*02, the only haplotype that differed significantly between patients and controls (one-way parametric Anova, F = 4.866, p = 0.028; OR = 7.33, p = 0.06), did not exhibit significant linkage disequilibrium. CONCLUSION: These results show that HLA-DRB1*07, associated with RHD in various world populations, is also associated with RHD in the Pakistani population. The validation of HLA associations with RHD, which is observed in different world populations, may lead to the development of a cost-effective strategy in the primary prevention of this disease.     

Inhibition of ovarian cancer cell metastasis by a fusion polypeptide Tat-ELP

Tumor cell metastasis is a complex, multi-step process that is a major cause of death and morbidity amongst cancer patients. Cell adhesion plays a critical role in the development of metastatic cancer, and it is mediated by interactions between receptors on the cell surface and ligands of the extracellular matrix or other surfaces. Therefore, inhibition of the cell adhesion process appears to be an effective method of preventing metastasis. This work describes a genetically engineered polypeptide with the potential to prevent cell adhesion and inhibit metastasis. We have found that the cell penetrating peptide Tat, fused with elastin-like polypeptide (ELP) inhibited adhesion, spreading, invasion and migration of SKOV-3 ovarian cancer cells in cell culture. Furthermore, we have also confirmed that Tat-ELP has anti-metastatic potential in an experimental ovarian cancer metastasis model in vivo, causing approximately 80% reduction in the tumor burden. Since cell attachment is an important step in tumor cell invasion and metastasis, these results suggest a novel role of Tat-ELP as a therapeutic intervention in cancer metastasis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Surgical site infection rate and associated risk factors in elective general surgery at a public sector medical university in Pakistan

This prospective study aimed to determine the surgical site infection (SSI) rate and associated risk factors was carried in a general surgical ward at Liaquat University Hospital Jamshoro. A total of 460 patients requiring elective general surgery from July 2005 to June 2006 were included in this study. All four surgical wound categories were included. Primary closure was employed in all cases. Patients were followed up to 30th day postoperatively. All cases were evaluated for postoperative fever, redness, swelling of wound margins and collection of pus. Cultures were taken from all the cases with any of the above finding. Mean +/- SD age of the patients was 38.8 +/- 17.4 years with male to female ratio of 1.5:1. The overall rate of surgical site infection was 13.0%. The rate of wound infection was 5.3% in clean operations, 12.4% in clean-contaminated, 36.3% in contaminated and 40% in dirt-infected cases. Age, use of surgical drain, duration of operation and wound class were significant risk factors for increased surgical site infection (P < 0.05). Postoperative hospital stay was double in cases who had surgical site infection. Sex, haemoglobin level and diabetes were not statistically significant risk factors (P > 0.05). In conclusion, surgical site infection causes considerable morbidity and economic burden. The routine reporting of SSI rates stratified by potential risk factors associated with increased risk of infection is highly recommended.     

Immunosuppressive effects of mesenchymal stem cells: involvement of HLA-G

INTRODUCTION: Mesenchymal stem cells (MSCs) possess unique immunomodulatory properties. They are able to suppress allogenic T-cell response and modify maturation of antigen-presenting cells. Their role in the treatment of severe graft versus host disease has been reported. The underlying molecular mechanisms of immunosuppression are currently being investigated. Histocompatibility locus antigen (HLA)-G is a nonclassical major histocompatibility complex class I antigen with strong immune-inhibitory properties. METHODS: We studied the role of HLA-G on MSC-induced immunosuppression. The expression of HLA-G on human MSCs cultured alone and in mixed lymphocytes reaction (MSC/MLR) was analyzed. RESULTS: We found that HLA-G can be detected on MSCs by real-time reverse-phase polymerase chain reaction, immunofluorescence, flow cytometry (52.4+/-3.6%), and enzyme-linked immunosorbent assay in the supernatant (38.7+/-5.2 ng/mL). HLA-G protein expression is constitutive and the level is not modified upon stimulation by allogenic lymphocytes in MSC/MLR. The functional role of HLA-G protein expressed by MSCs was analyzed using the 87G anti-HLA-G blocking antibody in a MSC/MLR. We found that blocking HLA-G molecule significantly raised lymphocyte proliferation in MSC/MLR (35.5%, P=0.01). CONCLUSION: Our findings provide evidences supporting involvement of HLA-G in the immunosuppressive properties of MSCs. These results emphasize the potential application of MSCs as a relevant therapeutic candidate in transplantation.     

Increased incidence of mosaicism detected by FISH in murine blastocyst cultured in vitro

The majority of in-vitro-derived human preimplantation embryos are chromosomally abnormal but whether the same pattern exists in vivo is unknown. This would be impossible to demonstrate in humans. Hence we chose murine embryos to study this difference owing to their ease of manipulation and compared the incidence of mosaicism between in-vivo- and in-vitro-cultured embryos. Two groups of embryos were analysed. Group A (in vitro) were obtained 48h following superovulation and cultured in vitro until the blastocyst stage. Fluorescent in-situ hybridization (FISH) was performed at different stages that included the cleavage, morula and blastocyst stage. Group B (in vivo) were obtained on day 2 or day 5 and FISH was performed immediately without culture. There was an increase in chromosomal mosaicism seen from the cleavage stage up to the blastocyst stage in the in-vitro culture group. Overall chromosomal abnormality from day 3 to day 5 was found to be 30% (28/94) in group A. The incidence of chromosomal abnormalities in blastocysts from group B was significantly lower than group A blastocysts (8% (3/40) and 31% (20/64) respectively; P<0.05). These data show that in-vitro cultured embryos had a significantly higher incidence of mosaicisim in comparison with the in-vivo group. Cultured human embryos show high levels of chromosomal abnormalities but whether this is a pattern seen in all embryos or is the result of culture is unknown. To study this pattern we used mouse embryos and carried out chromosome analysis by fluorescent in-situ hybridization. We compared embryos that were cultured (in vitro) with those that were not (in vivo, i.e. grown exclusively in the mouse). We found that cultured embryos showed significantly higher chromosomal abnormalities as compared with in vivo embryos. This suggests that certain culture conditions are responsible for the high level of chromosomal abnormalities seen in these embryos, which should be investigated further.