Indication of an involvement of interleukin-1 beta converting enzyme- like protease in intramedullary apoptotic cell death in the bone marrow of patients with myelodysplastic syndromes

Our previous studies using in situ end labeling (ISEL) of fragmented DNA revealed extensive apoptotic cell death in the bone marrows (BM) of patients with myelodysplastic syndromes (MDS) involving both stromal and hematopoietic cells. In the present report we show greater synthesis of interleukin-1 beta (IL-1 beta) in 4 hour cultures of density separated BM aspirate mononuclear (BMAM) cells from MDS patients as compared to the cultures of normal BM from healthy donors or lymphoma patients (1.7 +/- 0.37 pg/10(5) cells, n = 29 v 0.42 +/- 0.24 pg/10(5) cells, n = 11, respectively, P = .049). Further, these amounts of IL-1 beta in MDS showed a significant correlation with the extent of apoptosis detected by ISEL in corresponding plastic embedded BM biopsies (r = .480, n = 30, P = .007). In contrast normal BMs did not show any correlation between the two (r = .091, n = 12, P = .779). No significant correlation was found between the amounts of IL-1 beta and % S-phase cells (labeling index; LI%) in MDS determined in BM biopsies using immunohistochemistry following in vivo infusions of iodo- and/or bromodeoxyuridine. Neither anti-IL-1 beta antibody nor IL-1 receptor antagonist blocked the apoptotic death of BMAM cells in 4 hour cultures (n = 5) determined by ISEL (apoptotic index; AI%), although the latter led to a dose-dependent accumulation of active IL-1 beta in the culture supernatants. On the other hand, a specific tetrapetide- aldehyde inhibitor of ICE significantly retarded the apoptotic death of BMAM cells at 1 mumol/L in 5/6 MDS cases studied (AI% = 2.99 +/- 0.30 in controls v 1.58 +/- 0.40 with ICE-inhibitor, P = .05) and also reduced the levels of active IL-1 beta synthesized (5.59 +/- 2.63 v 2.24 +/- 0.93 pg/10(6) cells, respectively). In normal cells, neither IL-1 blockers nor the ICE inhibitor showed any effect on the marginal increase in apoptosis observed in 4 hour cultures. Our data thus suggest a possible involvement of an ICE-like protease in the intramedullary apoptotic cell death in the BMs of MDS patients.     

Low-temperature storage of bone marrow in nitrogen vapor-phase refrigerators: decreased temperature gradients with an aluminum racking system

Large temperature gradients may exist in nitrogen vapor-phase refrigerators. Cryopreserved cells stored at higher levels may be considerably warmer than those stored closer to the liquid nitrogen reservoir. To decrease this temperature gradient, racking systems constructed of aluminum were placed in marrow storage refrigerators. The higher heat conductivity of aluminum resulted in a vapor-phase temperature gradient of only 5.9 degrees C at 22.5 inches above the liquid, as compared to the gradient of 86 degrees C seen with steel frames in a similar refrigerator. Temperature fluxes were minimal with lid opening or nitrogen filling. The thicker frame size and loss of the lowest storage level resulted in a storage capacity 63 percent of that achievable with steel frames and liquid immersion. Consumption of nitrogen was estimated to be 174 to 220 percent of the static usage in this model of refrigerator with 6 inches of nitrogen, but comparable to the consumption expected with full immersion of the racking system, regardless of frame construction. These data demonstrate the feasibility of achieving very low, stable, cryogenic temperatures in a vapor-phase refrigerator.     

Clostridium difficile after haemolytic uraemic syndrome

Six children are described who developed diarrhoea associated with Clostridium difficile during the course of haemolytic uraemic syndrome. The significance of this infection is discussed within the context of the pathophysiology of haemolytic uraemic syndrome.     

Invariant Natural Killer T-Cell Control of Type 1 Diabetes: A Dendritic Cell Genetic Decision of a Silver Bullet or Russian Roulette

OBJECTIVE

In part, activation of invariant natural killer T (iNKT)-cells with the superagonist α-galactosylceramide (α-GalCer) inhibits the development of T-cell–mediated autoimmune type 1 diabetes in NOD mice by inducing the downstream differentiation of antigen-presenting dendritic cells (DCs) to an immunotolerogenic state. However, in other systems iNKT-cell activation has an adjuvant-like effect that enhances rather than suppresses various immunological responses. Thus, we tested whether in some circumstances genetic variation would enable activated iNKT-cells to support rather than inhibit type 1 diabetes development.

RESEARCH DESIGN AND METHODS

We tested whether iNKT-conditioned DCs in NOD mice and a major histocompatibility complex–matched C57BL/6 (B6) background congenic stock differed in capacity to inhibit type 1 diabetes induced by the adoptive transfer of pathogenic AI4 CD8 T-cells.

RESULTS

Unlike those of NOD origin, iNKT-conditioned DCs in the B6 background stock matured to a state that actually supported rather than inhibited AI4 T-cell–induced type 1 diabetes. The induction of a differing activity pattern of T-cell costimulatory molecules varying in capacity to override programmed death-ligand-1 inhibitory effects contributes to the respective ability of iNKT-conditioned DCs in NOD and B6 background mice to inhibit or support type 1 diabetes development. Genetic differences inherent to both iNKT-cells and DCs contribute to their varying interactions in NOD and B6.H2^g7 mice.

CONCLUSIONS

This great variability in the interactions between iNKT-cells and DCs in two inbred mouse strains should raise a cautionary note about considering manipulation of this axis as a potential type 1 diabetes prevention therapy in genetically heterogeneous humans.Invariant natural killer T (iNKT)-cells are a small regulatory lymphocyte subset characterized by their unique ability to recognize glycolipid antigens presented by the major histocompatibility complex (MHC) class I–like CD1d molecule (1). Activated iNKT-cells induce profound multiple effects on innate and adaptive immune responses, primarily through rapid secretion of various cytokines and other agents (1,2). Among the downstream events regulated by activated iNKT-cells is the maturation of dendritic cells (DCs) that subsequently induce various adaptive immune responses (1). Activating iNKT-cells with various agonists has shown promise in modulating DC functions for both stimulating immunological responses against tumors and infectious agents as well as inducing tolerogenic responses for inhibiting autoimmune syndromes (1,3,4). Unfortunately, in regard to possible iNKT-mediated therapeutic approaches in humans, too little is currently known about how iNKT-cells promote differentiation of immunogenic versus tolerogenic DCs. Without an ability to regulate desired downstream DC responses, the danger exists of iNKT-directed protocols exacerbating the disease being treated (3,4). Therefore, much attention is currently focused on characterizing particular courses of iNKT-cell–induced DC differentiation.Mouse-based studies assessing the ability of iNKT-cells activated by the superagonist α-galactosylceramide (α-GalCer) to enhance antitumor immunity have almost exclusively used the C57BL/6J (B6) or BALB/c strains (5–10). More strains have been evaluated for α-GalCer–induced suppression of various autoimmune syndromes (4). Hence, strain-dependent factors may be important in determining whether activated iNKT-cells induce immunogenic or tolerogenic events. Indeed, there is great strain variability in iNKT-cell numbers that also differ in cytokine production profiles (11). However, it remains unknown to what extent genetic variability may determine whether activated iNKT-cells subsequently induce immunogenic versus tolerogenic differentiation of DCs.The ability of α-GalCer–activated iNKT-cells to inhibit autoimmune type 1 diabetes development in NOD mice may be at least partly due to downstream maturation of tolerogenic DCs (2). iNKT-conditioned DCs in NOD mice preferentially accumulated in pancreatic lymph nodes where some diabetogenic T-cells subsequently underwent apoptotic deletion, but with a larger proportion becoming functionally anergized (2). However, before α-GalCer–based type 1 diabetes intervention approaches could be considered in humans, it would be important to know whether patient genetic heterogeneity may result in variable types of downstream DC responses. Indeed, in the current study, we found that unlike in NOD mice, in a B6 background stock iNKT-conditioned DCs not only failed to mature to a type 1 diabetes–protective state, but actually support disease development due to differing expression of various T-cell costimulatory and inhibitory molecules.     

A study of the knowledge and attitude towards pulse polio immunization in semi urban areas of South India

Background

The government of India launched the pulse polio immunization (PPI) programme in 1995 with the aim of eradicating poliomyelitis by the end of 2000. Despite this, 733 children with polio were reported in 2009 alone. Therefore, there is a need to understand the reason underlying such high numbers of cases after so many years of programme implementation. This study was performed to assess the knowledge of the general population about poliomyelitis and PPI and their attitude and practice towards PPI.

Method

This cross-sectional study was undertaken in two semi- urban areas of Mangalore city. Only houses in which children under five lived were included in the study. Data was collected by interviewing any adult member of the household using a pretested questionnaire.

Results

The literacy rate of study participants was 99%. Only 35(10.9%) participants knew the correct mode of transmission of polio. More than one quarter of the study population were under the misconception that polio is a curable disease. The primary source of information about PPI in majority of participants was the television (n = 192; 60%). Two-hundred and eighty eight (90%) participants knew that the purpose of PPI was to eradicate polio. Only 128 (40%) participants knew that polio drops can be given to children with mild illnesses and an identical number of participants knew that hot food stuff should not be given for at least half an hour following vaccination administration. Misconceptions such as PPI causing vaccine overdose was identified among 7 (2.2%) participants, it is a substitute for routine immunization was believed among 30 (9.4%) participants and that oral polio vaccine prevents other diseases was seen among 76 (23.7%) participants. The educational status of the participants was significantly associated with their awareness level (χ² =13.668, DF=6, P=0.033).

Conclusion

This study identified a few important misconceptions associated with polio and PPI which need to be addressed by large scale awareness campaigns in order to achieve polio eradication in the near future.