Establishment of transplantation tolerance via minimal conditioning in aged recipients.

Mixed hematopoietic chimerism is a powerful means of generating donor-specific tolerance, allowing long-term graft acceptance without lifelong dependence on immunosuppressive drugs. To avoid the need for whole body irradiation and associated side effects, we utilized a radiation-free minimal conditioning regime to induce long-term tolerance across major histocompatibility barriers. We found that low-dose busulfan, in combination with host T cell depletion and short-term sirolimus-based immunosuppression, facilitated efficient donor engraftment. Tolerance was achieved when mice were transplanted with whole or T cell-depleted bone marrow, or purified progenitor cells. Tolerance induction was associated with an expansion in regulatory T cells and was not abrogated in the absence of a thymus, suggesting a dominant or compensatory peripheral mode of tolerance. Importantly, we were able to generate durable chimerism and tolerance to donor skin grafts in both young and aged mice, despite age-related thymic atrophy and immune senescence. Clinically, this is especially relevant as the majority of transplant recipients are older patients whose immune recovery might be dangerously slow and would benefit from radiation-free minimal conditioning regimes that allow efficient donor engraftment without fully ablating the recipient immune system.

Coagulation patterns following haemoglobin-based oxygen carrier resuscitation in severe uncontrolled haemorrhagic shock in swine.

Massive blood loss due to penetrating trauma and internal organ damage can cause severe haemorrhagic shock (HS), leading to a severely compromised haemostatic balance. This study evaluated the effect of bovine polymerized haemoglobin (Hb) (Hb-based oxygen carrier, HBOC) resuscitation on haemostasis in a swine model of uncontrolled HS. Following liver injury/HS, swine received HBOC (n= 8), Hextend (HEX) (n= 8) or no resuscitation (NON) (n= 8). Fluids were infused to increase mean arterial pressure above 60 mmHg and to reduce heart rate to baseline. At 4 h, the animals were eligible for blood transfusions. Prothrombin time (PT), activated partial thromboplastin time, fibrinogen, thromboelastography (TEG) and platelet function analyser closure time (PFA-CT) were compared by using mixed statistical model. At 4 h, blood loss (% estimated blood volume) was comparable for HBOC (65.5 +/- 18.5%) and HEX (80.8 +/- 14.4%) and less for NON (58.7 +/- 10.1%; P < 0.05). Resuscitation-induced dilutional coagulopathy was observed with HBOC and HEX, as indicated by reduced haematocrit, platelets and fibrinogen (P < 0.05). At 4 h, PT was higher in HEX than in HBOC groups (P < 0.01). In the early hospital phase, a trend to increased TEG reaction time and PFA-CT indicates that dilutional effects persist in HBOC and HEX groups. PFA-CT returned to baseline later with HBOC than with HEX (48 vs. 24 h) following blood transfusion. At 4 h, all surviving HEX animals (n= 3) required transfusion, in contrast to no HBOC (n= 7) or NON (n= 1) animals. In this severe uncontrolled HS model, successful resuscitation with HBOC produced haemodilutional coagulopathy less than or similar to that produced by resuscitation with HEX.

Improved detection of clinically significant host-reactive antigens prior to HLA-identical sibling peripheral blood stem cell transplantation using a dendritic cell-based helper T-lymphocyte precursor assay.

SUMMARY: Graft-versus-host disease (GVHD) due to host-reactive antigenic differences between HLA-identical pairs remains an important cause of morbidity and mortality after allogeneic transplantation. The helper T-lymphocyte precursor (HTLp) assay, using peripheral blood mononuclear cells (PBMCs), has been variably shown to detect such host-reactive differences. We assessed whether using dendritic cells (DCs) as the stimulator cells would improve the ability of the HTLp assay to detect these differences. We used PBMCs (standard HTLp assay) or monocyte-derived DCs (DC-HTLp assay) as the stimulator cells for 12 HLA-identical sibling pairs undergoing allogeneic peripheral blood stem cell transplantation. HTLp frequencies were greater by the DC-HTLp assay (median 1:77 712 vs 1:727 514; P=0.008). The standard HTLp assay did not predict for acute GVHD (P=0.42), whereas a trend was noted for the DC-HTLp assay (P=0.095). Of note, of seven patients developing moderately severe to severe acute GVHD, four had a significantly greater DC-HTLp frequency compared to the standard HTLp frequency, whereas all four patients who developed no to moderate acute GVHD had similar HTLp frequencies whether PBMCs or DCs were used as the stimulator cells. Although the small number of donor/recipient pairs assessed limits the strength of any conclusions, our study suggests that the DC-HTLp assay is better able to detect clinically significant host-reactive antigenic differences between HLA-identical siblings.

Fas and Fas ligand expression on human peripheral blood leukocytes.

OBJECTIVES: Study of Fas and Fas ligand (Fas-L) expression, as well as sFas-L release, by fresh human peripheral blood leukocytes. METHODS: Flow cytometry, cytotoxicity, immunofluorescence staining of fresh smears. Western blotting. RESULTS: Granulocytes and monocytes express a low level of Fas receptor, but no Fas-L. These cells, as well as NK cells, contain presynthesized depots of Fas-L which they express following activation by brief storage (60 min) at room temperature or during separation from whole blood. Such activation also leads to Fas receptor upregulation. NK cells do not express Fas receptor. Once expressed on blood leukocytes, fully functional Fas-L can be released from the membrane and can be detected in plasma-free cell supernatants. CONCLUSION: Human peripheral blood granulocytes, monocytes and NK cells contain intracellular presynthesized Fas-L which they readily express following blood anticoagulation, blood storage or cell separation. Soluble Fas-L is released from those cells and can be detected in protein-free supernatants by immunoblotting.

Homicide surveillance--United States, 1979-1988.

From 1979 through 1988, 217,578 homicides occurred in the United States, an average of greater than 21,000 per year. Homicide rates during this 10-year period were about 1.5 times higher than the rates during the 1950s. The national homicide rate of 10.7/100,000 in 1980 was the highest ever recorded. Homicide occurs disproportionately among young adults. Among the 15- to 34-year age group, homicide is the fourth most common cause of death among white females, the third most common cause among white males, and the most common cause among both black females and black males. In 1988, nearly two-thirds (61%) of homicide victims were killed with a firearm, 75% of these with a handgun. More than half (52%) of homicide victims were killed by a family member or acquaintance, and about one-third (35%) of homicides stemmed from a conflict not associated with another felony. The homicide mortality rate among young black males 15-24 years of age has risen 54% since 1985. Ninety-nine percent of the increase was accounted for by homicides in which the victim was killed with a firearm. The surveillance data summarized in this report should assist public health practitioners, researchers, and policymakers in addressing this important public health problem.

Multifaceted freezing injury in human polymorphonuclear cells at high subfreezing temperatures.

Extracellular freezing injury at high subzero temperatures in human polymorphonuclear cells (PMNs) was studied with a cryomicroscope, electron microscope, and functional assays (phagocytosis, microbicidal activity, and chemotaxis). There are at least four major factors in freezing injury: osmotic stress, chilling, cold shock, and dilution shock. Extracellularly frozen PMNs lose functions when cooled to -2 degrees C without a cryoprotectant. Cells lose volume on freezing to the same degree as in hypertonic exposure. PMNs have a minimum volume to which they can shrink without injury. Greater dehydration produces irreversible injury to cellular functions, and cells eventually collapse under high osmotic stress. Chilling sensitivity is seen in slowly chilled, supercooled PMNs below -5 degrees C; at -7 degrees C, functions are lost in 1 h. This injury can be prevented by the addition of Me2SO but not glycerol. Me2SO does not, however, prevent cold shock (injury due to rapid cooling), which is seen during cooling at 10 degrees C/min to -14 degrees C, but not during slow cooling at 0.5 degrees C/min. One of the problems of using glycerol as a cryoprotectant stems from the high sensitivity of PMNs to dilution shock during the dilution or removal of glycerol.

5-L-hydroxy-6,8,11,14-eicosatetraenoate potentiates the human neutrophil degranulating action of platelet-activating factor.

Platelet-activating factor (AAGPC) and two of its structural analogues degranulated human neutrophils with respective potencies that were increased up to 100 to 1000-fold by 16 nM to 5 microM of 5-L-hydroxyeicosatetraenoate (5-L-HETE). 5-rac-HETE had similar actions but 8-rac-HETE was without effect. Furthermore, 5-L-HETE did not influence the degranulating actions of C5a, A23187 or a formalated oligopeptide chemotactic factor and none of the HETEs, by themselves, caused degranulation. Thus, 5-L-HETE and AAGPC selectively interact to induce degranulation. Since these products rapidly form in stimulated PMNs, they may serve as potentiator and agonist, respectively, to transduce biological signals into cell function.

Platelet-activating factor and analogues: comparative studies with human neutrophils and rabbit platelets.

Platelet-activating factor and 12 structural analogues stimulated rabbit platelets to aggregate and release [14C]-serotonin. They likewise caused human neutrophils to aggregate, degranulate, and take up [3H]-deoxyglucose. Their respective potencies, which varied by 4-5 orders of magnitude, correlated highly (r greater than or equal to 0.93) in all assays. These compounds also selectively desensitized neutrophils to the degranulating actions of platelet-activating factor but not to C5a or a formylated oligopeptide. Three other analogues with structures quite similar to platelet-activating factor were unable to activate or desensitize the cells. Hence, the structure-activity relations of the analogues in several assays of platelet and neutrophil function were similar and they stimulated neutrophils by a common activation mechanism that differed from those used by C5a or formylated oligopeptides. These data are consistent with the notion that platelet-activating factor activates and desensitizes various target cells through stereospecific receptors. Apparently, these putative receptors on neutrophils and platelets have similar structural specificities for platelet-activating factor and its analogues.