Effects on Hedonic Feeding, Energy Expenditure and Balance of the Non-opioid Peptide DYN-A2-17.

The dynorphin (DYN) peptide family includes opioid and non-opioid peptides, yet the physiological role of the non-opioid DYN peptides remains poorly understood. Recent evidence shows that administering the non-opioid peptide DYN-A2-17 into the paraventricular hypothalamic nucleus (PVN) simultaneously increased short-term intake of standard rodent chow and spontaneous physical activity (SPA). The present studies aimed to expand upon the mechanisms and role of DYN-A2-17 on food intake and energy expenditure. Injection of DYN-A2-17 in PVN increased SPA, energy expenditure and wheel running in the absence of food. Repeated DYN-A2-17 injection in PVN increased short-term chow intake, but this effect habituated over time and failed to alter cumulative food intake, body weight or adiposity. Pre-treatment with a CRF receptor antagonist into PVN blocked the effects of DYN-A2-17 on food intake while injection of DYN-A2-17 in PVN increased plasma ACTH. Finally, as DYN peptides are co-released with orexin peptides, we compared the effects of DYN-A2-17 to orexin-A and the opioid peptide DYN-A1-13 on food choice and intake in PVN when palatable snacks and chow were available. DYN-A1-13 selectively increased intake of palatable snacks. DYN-A2-17 and orexin-A decreased palatable snack intake while orexin-A also increased chow intake. These findings demonstrate that the non-opioid peptide DYN-A2-17 acutely regulates physical activity, energy expenditure and food intake without long-term effects on energy balance. These data also propose different roles of opioid, non-opioid DYN and orexin peptides on food choice and intake when palatable and non-palatable food options are available.

Correction of defective expression in MHC class II deficiency (bare lymphocyte syndrome) cells by retroviral transduction of CIITA.

Retrovirus-mediated gene transfer was used to restore expression to MHC class II-negative patient cells from complementation group A(II) of MHC class II immunodeficiency or bare lymphocyte syndrome (BLS). The cells of these patients do not transcribe MHC class II genes due to a defect in the trans-acting factor, CIITA. We constructed a vector, pGAG/Ii-CIITA, with the MHC class II-associated invariant chain promoter driving CIITA expression. Cocultivation with the virus producer line was consistently shown to be the optimal method for infection of all cell types. The induction of MHC class II expression after virus infection was rapid, and high levels of expression were achieved in cell lines within 1 wk of infection. In addition, expression was easily detectable even in peripheral blood cells of a BLS patient within a few days. Cell lines maintained in vitro for several months remained positive, and the proportion of cells with surface expression of DR was correlated with the number of integrated proviruses. Moreover, transduced B lymphoblastoid cell lines readily established tumors in CB17-scid/scid mice, and the MHC class II-positive cells demonstrated a clear competitive advantage in vivo. Ultimately, we hope to use this transduction system to restore normal immune function to a BLS patient for which no other therapeutic option currently exists.

Multiple minor histocompatibility antigen-specific cytotoxic T lymphocyte clones can be generated during graft rejection after HLA-identical bone marrow transplantation.

Graft rejection after T cell-depleted HLA-genotypically identical bone marrow transplantation (BMT) is probably mediated by mH antigen-specific cytotoxic T lymphocytes (CTL). We have analyzed peripheral blood mononuclear cells (PBMC) from a female bone marrow graft recipient, collected during graft rejection after a sex mismatched HLA-identical BMT. A CTL line was generated by stimulating recipient PBMC collected during graft rejection with donor PBMC and donor EBV-transformed lymphoblastoid cell lines. From this CTL line a large number of clones of different specificity and phenotype was established by limiting dilution. These clones exhibited several mH antigen specificities, restricted by HLA-B7, -B27 or -DR2 as shown by differential recognition of family members and unrelated individuals sharing potential restriction elements. The CD3+CD4+ and CD3+CD8+ bulk culture was cloned, resulting in 50 HLA-B7 restricted CD3+CD4-CD8+CTL clones, three HLA-B27 restricted CD3+CD4-CD8+CTL clones, one HLA-DR2 restricted CD3+CD4+CD8-CTL clone and two additional HLA class II restricted CD3+CD4+CD8-CTL clones with a different specificity. One representative clone of each specificity was selected for further analysis. The CTL line and the HLA-B7 restricted CD8+CTL clone, but not the HLA class II restricted CD4+ CTL clone, inhibited the growth of donor hematopoietic progenitor cells (HPC). In conclusion, these results show that graft rejection after HLA-identical BMT may be mediated by multiple CTL clones that specifically recognize one mH antigen peptide presented by different HLA molecules or different mH antigens expressed on donor cells and that CTL, but not CD4+ CTL inhibited donor HPC growth.

Serum granulocyte colony-stimulating factor (G-CSF) levels after allogeneic T cell-depleted marrow transplantation.

Endogenously produced and exogenously administered granulocyte colony-stimulating factor (G-CSF) has correlated with myeloid engraftment in a number of hematopoietic progenitor cell transplantation settings. Given the increased susceptibility of T cell-depleted (TCD) bone marrow transplants (BMT) to graft failure, a cohort of 36 (21 male and 15 female) recipients of TCD BMT was evaluated prospectively during the first month post-transplant for circulating serum G-CSF levels, to examine the correlation between myeloid engraftment and G-CSF levels. All recipients of TCD BM had measurable G-CSF levels, with a median peak level of 1750 pg/ml (range 540-26,250 pg/ml) occurring at a median of 5 days (range 1-18 days) after BM infusion. There was no association between G-CSF kinetics within 1 month post-transplant and the development of primary non-engraftment or secondary graft failure. One patient with primary non-engraftment and 6 patients with secondary graft failure exhibited median G-CSF peak levels of 1600 pg/ml and 1850 pg/ml (range 600-16,250 pg/ml) occurring 5 and 5.5 days (range 4-7 days) after BM infusion, respectively. Additionally, the patient with primary non-engraftment demonstrated a high G-CSF level in response to a low absolute neutrophil count (ANC). An inverse relationship between serial G-CSF levels and concomitant ANC was documented (log G-CSF = 6.19-0.009 ANC, P < 0.001). Higher peak G-CSF levels were associated with older recipient age (P = 0.01) and lower BM cell dose (P = 0.02), while administration of anti-thymocyte globulin post-transplant did not alter G-CSF levels.

Serum interleukin-3 levels following autologous or allogeneic bone marrow transplantation: effects of T-cell depletion, blood stem cell infusion, and hematopoietic growth factor treatment.

Engraftment of marrow following autologous or allogeneic bone marrow transplantation (BMT) may be influenced by quantity and function of stem cells. T lymphocytes, supporting microenvironmental cells, and hematopoietic growth factors (HGF). To elucidate the physiologic role of interleukin-3 (IL-3) in the engraftment process, serum IL-3 levels were measured in over 400 samples from 77 transplant recipients before and for up to 3 weeks following transplantation using a novel enzyme-linked immunoabsorbent assay (ELISA) with a sensitivity of > or = 78 pg/mL. Thirty-seven patients received two to three log T-cell-depleted allografts. In the remaining 40 patients (18 autologous marrow, 12 allogeneic marrow, and 10 autologous peripheral blood [PB] stem cell), T cells were not depleted (non-TCD) from the grafts. A burst of IL-3 (peak levels, 1,500 to 6,000 pg/mL) was detected in the immediate posttransplant period between day 0 and day 14 in all non-TCD recipients and in 21 of 37 (57%) of TCD recipients. A strong inverse relationship between IL-3 levels and absolute neutrophil count (ANC) was observed in both non-TCD recipients (r = -.796) and in TCD recipients (r = -.897). However, both peak IL-3 levels and mean IL-3 levels from day 0 through 14 were significantly lower in TCD recipients compared with either autologous or unmodified allogeneic marrow recipients (P < .01). The lowest peak or mean day 0 through 14 IL-3 levels were observed in matched related recipients undergoing the most aggressive (2.5 to 3.0 log) T-cell-depleted BMT. Autografted patients receiving blood stem cell transplants alone or posttransplant granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF) also had significantly lower peak IL-3 levels (P < .01). In patients receiving TCD grafts, administration of antithymocyte globulin (ATG) posttransplant significantly increased peak IL-3 levels compared with patients not treated with ATG (P < .04). This study shows that endogenous release of IL-3 is strongly associated with myeloid engraftment and inversely related to ANC. Removal of T lymphocytes from donor marrow or acceleration of engraftment by use of stem cells or growth factors appears to blunt the endogenous release of IL-3 whereas use of ATG posttransplant increases IL-3 release.

The effects of dehydration on the dynamics of transcapillary refill.

Since the water reserve of the interstitium plays a major role in circulatory homeostasis, its reduction by dehydration may produce severe changes in the organism's response to hemorrhage but this has not been measured experimentally. Twelve immature pigs (22 +/= 2 Kg) were divided into two groups of six each. Control animals had free access to food and water prior to bleeding. Dehydrated animals had water withheld for 48 hours preceding the bleeding. All animals were bled 30 per cent of their calculated blood volumes while awake. No resuscitation was performed. No mortality was observed in the control group of animals, while four of the six dehydrated animals died (66%). All deaths occurred between one and four hours posthemorrhage. Plasma refill reached 33 per cent by 0.5 hours in the control group compared to only 17 per cent by 0.5 hours in the dehydrated group (p less than or equal to .05). Refill in the control group reached 50 per cent by three hours, whereas dehydrated animals surviving to three hours demonstrated no further refill (p less than or equal to .05). BUN, calcium, sodium, and osmolality were consistently higher in dehydrated than control animals (p less than or equal to .05). It is concluded that a reduction in the interstitial water reserve significantly impairs ability to recover from hemorrhage.