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3.12 Acute stress impairs lymphocyte function but spares pancreatic endocrine function
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Cell Transplantation • Volume 5, Number 5S-2, 1996
3.11 CHRONIC HIGH GLUCOSE IMPAIRS INSULIN PROCESSING IN HUMAN ISLETS. Bertuzzl F, Saccomanno K, Socci C, Berra C, Taglietti MV, Dalcin E, Pozza G and Pontiroli AE; Milan ITALY.
3.12
Acute hyperthermic stress induces the synthesis of the heat shock proteins (HSPs). Maximal expression of HSPs occurs 8-16 hours following hyperthermia [heat shock (HS), 42.5°C x 15 minutes] and is associated with protection of pancreatic islets from warm ischemic injury. Islets isolated from these HS pancreata successfully reversed streptozotocin-induced hyperglycemia (Trans Proc 26(6):3477,1994). Acute inflammatory stress is associated with a generalized inhibition of protein synthesis in the islet cell (Autoimraunity 9:33,1991). We wished to: A) determine if HS causes early islet dysfunction and B) compare islet and T-lymphocyte function in the same animal following HS. Methods: Islets and T-lympbocytes were isolated from male Lewis donor rats. Heat pretreatments consisted of whole-body HS. Following 0.5, 8, 24, and 48 hours recovery, pancreatic islets and splenocytes were isolated from the same animal and their function compared with and without HS. Islet (insulin release; static incubation) and T-cell (IL-2 release; proliferation assay) functions were tested by in vitro stimulation with glucose/theophylline or mitogen, respectively. Sham heat treated and normal animals served as controls. Results: Pancreatic islets from HS rats displayed intact insulin responses to glucose and theophylline [stimulation indices (SI) = 1.21-7.33, n=6] that were not statistically different from the control groups (SI= 1.17-5.73, n=6). Conversely, lymphocytes from the same HS donors had significantly reduced IL-2 production compared to normal controls (24 hrs; 22% of controls, p=0.009). Islets and Tlymphocytes from HS donors expressed high levels of HSPs compared to controls. Conclusion: Following acute thermal stress in the rodent, a selective inhibition of cellular function was observed in T-lymphocytes while sparing pancreatic islet cells. These results suggest there exists organ specific differences in the outcome of heat shock gene expression.
Hyperpminsulinemia could be a secondary effect of increased secretory demand on B cells correlated with hyperglycemia. The aim of this study was to assess the effects of a culture in high glucose concentrations on islets content and release of insulin (I) and pminsulin-like molecules (PLM). Human islets from 6 pancreas after overnight culture in standard CMRL 1066, were cultured for 48-h in 5.5 mM or 16.7 mM glucose. After this period, islets were perifused and acutely stimulated for 20 minutes with glucose 16.7 mM, followed by glucose 3.3 mM for 20 min. As expected, islets cultured in high glucose lost the ability to release I in response to an acute glucose stimulation, and showed a paradoxical I response to low glucose levels. The PLM/I ratio was higher in islets cultured in high glucose than in control islets, after high glucose (0.47 + 0.10 vs 0.234- .04 of control islets; n=12; p<0.05), and low glucose (0.92 4- 0.24 vs 0.24+ .04 of control islets; n=8; p<0.05). By electron microscopy a rnorphologic analysis showed an increased ratio of PI to Icontaining granules (0.4+0.04 in control islets and 1.2.t:0.19 in test islets; p<0.01). Morphological analysis were confirmed by a double gold immunolabelling using specific moncclonal antibodies against pro-insulin (PI) or insulin (I). The evidence that culture conditions increase PLM release in isolated human islets suggests that a chronic glucose stimulation can imbalance the rates of I processing in B cell, so that insulin is released before its maturation is complete.
3.13
INTERACTION BETWEEN HUMAN HUMORAL FACTORS AND SWINE BONE MARROW CELLS (AN EXPERIMENTAL REPORT). de Di Risio CB, Barbich M, Murua A, Hyon SH, BeveraggJ MI, Argibay P; Buenos Aires ARGENTINA Swine bone marrow (SBM) transplantation could be useful in the development of tolerance in humans towards organs from swine and also in the treatment of AIDS patients. However, interactions between a human microenvimnment and SBM need to be proved in several ex-vivo assays. Our laboralory is investigating several culture asseo,s with xenogeneic combinations. One interesting experiment was effectuated culturing SBM, conditioned medium from human lymphocytes and human erythropoyetin. As a control we cultured SBM and conditioned medium from swine lymphocytes. Bone marrow was harvested from a landrac¢ pig through a sternal punction and depleted of red blood cells. One week cultures (200,000 cells/plate) were initiated in 35x10mm plates with culture medium in mcthylcellulose and conditioned medium from the culture of 2x106 PHA stimulated cells. The cultures were incubated at 37° C with 5% CO~. All the cells were harvested and screened for colonies (>40 cells). In the SBM/human conditioned medium combination, a mean growth of 150 colonieswas observed. With the addition of buman eq~ropoyetin we obtained 300 eo~lzroid bursts. In the control setting (SBM/swine conditioned medium combination) 169 colonies were obtained. Despite this being a very pmliminmy report, we think that there could be nospecies-specific restriction in the interaction between human humoral factors and swine bone marrow under these specific ex-vivo conditions.
ACUTE STRESS IMPAIRS LYMPHOCYTE FUNCTION BUT SPARES PANCREATIC ENDOCRINE FUNCTION. Rewinski MJ, Krastins M, Schweizer RT, Perdrizet GA; Hartford, USA
3.14
MONOCLONAL ANTIBODIES TO THE CELL SURFACE OF MCRI-39 MARK THIS HUMAN LYMPHOID/MYELOID CELL LINE BUT NOT HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS ~ o G, Russo DA, Bash JA, Kraveka J, and August CS; Miami, USA In an attempt to characterize the cell surface of MCHRI-39 cell line, monoclonal antibodies were raised in mice. The cell line has a predominant "B" cell phenolype but also abl~antly expresses some "T" and Myeloid markers (ex.: CD15,38). Ninety four of one hundred thirty five (70%) of the hybridoma supematants reacted with surface markers on MCRI-39, using both flow cytometry and an ELISA to intact MCRI-39 cells. Among the ninety four positives, the most reacdve were tested on human peripheral blood mononuclear cells (PBMC). Three of twenty-six supematants tested positive on both PBMC and MCRI-39. One supematant reacted with lymphocytes, monocytes and granulocytes while the other two had reaaivities on lymphocytes only. The majority of supematants tested reacted with markers on the MCRI-39 cell line but not to cells ¢ii~!!aling in the peripheral blood. Experiments are underway to further characterize cell surface markers that are unique to MCRI-39.
Cell Transplantation • Volume 5, Number 5S-2, 1996
3.11 CHRONIC HIGH GLUCOSE IMPAIRS INSULIN PROCESSING IN HUMAN ISLETS. Bertuzzl F, Saccomanno K, Socci C, Berra C, Taglietti MV, Dalcin E, Pozza G and Pontiroli AE; Milan ITALY.
3.12
Acute hyperthermic stress induces the synthesis of the heat shock proteins (HSPs). Maximal expression of HSPs occurs 8-16 hours following hyperthermia [heat shock (HS), 42.5°C x 15 minutes] and is associated with protection of pancreatic islets from warm ischemic injury. Islets isolated from these HS pancreata successfully reversed streptozotocin-induced hyperglycemia (Trans Proc 26(6):3477,1994). Acute inflammatory stress is associated with a generalized inhibition of protein synthesis in the islet cell (Autoimraunity 9:33,1991). We wished to: A) determine if HS causes early islet dysfunction and B) compare islet and T-lymphocyte function in the same animal following HS. Methods: Islets and T-lympbocytes were isolated from male Lewis donor rats. Heat pretreatments consisted of whole-body HS. Following 0.5, 8, 24, and 48 hours recovery, pancreatic islets and splenocytes were isolated from the same animal and their function compared with and without HS. Islet (insulin release; static incubation) and T-cell (IL-2 release; proliferation assay) functions were tested by in vitro stimulation with glucose/theophylline or mitogen, respectively. Sham heat treated and normal animals served as controls. Results: Pancreatic islets from HS rats displayed intact insulin responses to glucose and theophylline [stimulation indices (SI) = 1.21-7.33, n=6] that were not statistically different from the control groups (SI= 1.17-5.73, n=6). Conversely, lymphocytes from the same HS donors had significantly reduced IL-2 production compared to normal controls (24 hrs; 22% of controls, p=0.009). Islets and Tlymphocytes from HS donors expressed high levels of HSPs compared to controls. Conclusion: Following acute thermal stress in the rodent, a selective inhibition of cellular function was observed in T-lymphocytes while sparing pancreatic islet cells. These results suggest there exists organ specific differences in the outcome of heat shock gene expression.
Hyperpminsulinemia could be a secondary effect of increased secretory demand on B cells correlated with hyperglycemia. The aim of this study was to assess the effects of a culture in high glucose concentrations on islets content and release of insulin (I) and pminsulin-like molecules (PLM). Human islets from 6 pancreas after overnight culture in standard CMRL 1066, were cultured for 48-h in 5.5 mM or 16.7 mM glucose. After this period, islets were perifused and acutely stimulated for 20 minutes with glucose 16.7 mM, followed by glucose 3.3 mM for 20 min. As expected, islets cultured in high glucose lost the ability to release I in response to an acute glucose stimulation, and showed a paradoxical I response to low glucose levels. The PLM/I ratio was higher in islets cultured in high glucose than in control islets, after high glucose (0.47 + 0.10 vs 0.234- .04 of control islets; n=12; p<0.05), and low glucose (0.92 4- 0.24 vs 0.24+ .04 of control islets; n=8; p<0.05). By electron microscopy a rnorphologic analysis showed an increased ratio of PI to Icontaining granules (0.4+0.04 in control islets and 1.2.t:0.19 in test islets; p<0.01). Morphological analysis were confirmed by a double gold immunolabelling using specific moncclonal antibodies against pro-insulin (PI) or insulin (I). The evidence that culture conditions increase PLM release in isolated human islets suggests that a chronic glucose stimulation can imbalance the rates of I processing in B cell, so that insulin is released before its maturation is complete.
3.13
INTERACTION BETWEEN HUMAN HUMORAL FACTORS AND SWINE BONE MARROW CELLS (AN EXPERIMENTAL REPORT). de Di Risio CB, Barbich M, Murua A, Hyon SH, BeveraggJ MI, Argibay P; Buenos Aires ARGENTINA Swine bone marrow (SBM) transplantation could be useful in the development of tolerance in humans towards organs from swine and also in the treatment of AIDS patients. However, interactions between a human microenvimnment and SBM need to be proved in several ex-vivo assays. Our laboralory is investigating several culture asseo,s with xenogeneic combinations. One interesting experiment was effectuated culturing SBM, conditioned medium from human lymphocytes and human erythropoyetin. As a control we cultured SBM and conditioned medium from swine lymphocytes. Bone marrow was harvested from a landrac¢ pig through a sternal punction and depleted of red blood cells. One week cultures (200,000 cells/plate) were initiated in 35x10mm plates with culture medium in mcthylcellulose and conditioned medium from the culture of 2x106 PHA stimulated cells. The cultures were incubated at 37° C with 5% CO~. All the cells were harvested and screened for colonies (>40 cells). In the SBM/human conditioned medium combination, a mean growth of 150 colonieswas observed. With the addition of buman eq~ropoyetin we obtained 300 eo~lzroid bursts. In the control setting (SBM/swine conditioned medium combination) 169 colonies were obtained. Despite this being a very pmliminmy report, we think that there could be nospecies-specific restriction in the interaction between human humoral factors and swine bone marrow under these specific ex-vivo conditions.
ACUTE STRESS IMPAIRS LYMPHOCYTE FUNCTION BUT SPARES PANCREATIC ENDOCRINE FUNCTION. Rewinski MJ, Krastins M, Schweizer RT, Perdrizet GA; Hartford, USA
3.14
MONOCLONAL ANTIBODIES TO THE CELL SURFACE OF MCRI-39 MARK THIS HUMAN LYMPHOID/MYELOID CELL LINE BUT NOT HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS ~ o G, Russo DA, Bash JA, Kraveka J, and August CS; Miami, USA In an attempt to characterize the cell surface of MCHRI-39 cell line, monoclonal antibodies were raised in mice. The cell line has a predominant "B" cell phenolype but also abl~antly expresses some "T" and Myeloid markers (ex.: CD15,38). Ninety four of one hundred thirty five (70%) of the hybridoma supematants reacted with surface markers on MCRI-39, using both flow cytometry and an ELISA to intact MCRI-39 cells. Among the ninety four positives, the most reacdve were tested on human peripheral blood mononuclear cells (PBMC). Three of twenty-six supematants tested positive on both PBMC and MCRI-39. One supematant reacted with lymphocytes, monocytes and granulocytes while the other two had reaaivities on lymphocytes only. The majority of supematants tested reacted with markers on the MCRI-39 cell line but not to cells ¢ii~!!aling in the peripheral blood. Experiments are underway to further characterize cell surface markers that are unique to MCRI-39.