Successful autografting in chronic myeloid leukaemia after maintenance of marrow in culture

We have previously shown that Philadelphia chromosome (Ph1)-positive cells rapidly disappear when marrow from patients with chronic myeloid leukaemia (CML) is cultured under conditions that maintain normal haematopoiesis for many weeks. The ability of marrow maintained in culture for 10 days to serve as an autograft has now been tested in three patients treated with intensive chemoradiotherapy. Two weeks after transplantation, marrow samples from all patients showed trilineage haematopoiesis. Neutrophil counts greater than 1.0 x 10(9)/l were achieved in all patients within 4 weeks, and platelet counts greater than 20 x 10(9)/l were achieved in two patients within 5 weeks. During this period of haematopoietic recovery, marrow cells were exclusively Ph1-negative in two patients and predominantly so in the third. These results suggest that engraftment can occur from Ph1-negative haematopoietic stem cells selected by maintenance of autologous CML marrow in culture for 10 days. Thus, the feasibility of using this approach to allow intensive and potentially curative therapy for CML has been established.     

Times for healing: towards a typology of time-frames in Swiss alcohol and drug clinics

AIM: To translate theoretical dimensions of 'social time' and 'clock time' in addiction treatment settings into empirical measures and to develop a typology of institutional time perspectives. METHOD: From November 2001 to February 2002, a mail survey was conducted with directors of 57 alcohol and drug clinics in the German-speaking part of Switzerland. Items measured the past and future orientation of the treatment programmes, elements of social time and clock time as part of organizational life and 'time bargaining' between therapists and clients. FINDINGS: Four clusters of temporal orientations emerged: 'clock time keepers' (who emphasize time control and future pessimism); 'nostalgic time riders' (focus on the 'good old days' but individualized planning); 'optimist speeders' (fast pacing, future control); and 'relaxed future optimists' (also future oriented but not sharing the idea of linear time). The time-frame also influences the negotiation of time in treatment between staff and patients: 'relaxed future optimists' most frequently report diverging views, while 'optimist speeders' seem to harmonize the views of patients and treatment staff more easily. Finally, the introduction of the harm reduction policy seems to covary with the ways in which time is viewed and used in treatment organizations. CONCLUSIONS: Treatment programmes differ in their view and use of time. Organizational times with varying dynamics, future-past orientations and time control interact with the organizational structure and socio-environmental factors. The capacity of programmes to adapt to changes in treatment policy and in the treatment system depends to some extent on organizational time orientation.     

Protein C Mangel — Risikofaktor für venöse Thrombosen

Summary Protein C is a vitamin K-dependent protein which is produced in the liver. Activated protein C has an anticoagulant effect by inactivating the clotting factors V and VIII. We report on a young female patient who suffered from recurrent thrombosis of the deep calf and pelvic veins with pulmonary embolism. Superficial thrombophlebitis also occurred repeatedly. In the plasma of this patient we found reduced levels of protein C antigen (0.62 U/ml) and activity (0.42 U/ml). Investigation of other family members revealed a protein C deficiency in her father, sister, and son. During the anticoagulant treatment with Marcumar the patient developed a coumarin-induced skin necrosis, to which complication a protein C deficiency is evidently predisposed. Thsi complication could only be prevented if heparin was given simultaneously during the adjustment period.

     

Umbilical cord mesenchymal stem cells: adjuvants for human cell transplantation.

The Wharton's jelly of the umbilical cord is rich in mesenchymal stem cells (UC-MSCs) that fulfill the criteria for MSCs. Here we describe a novel, simple method of obtaining and cryopreserving UC-MSCs by extracting the Wharton's jelly from a small piece of cord, followed by mincing the tissue and cryopreserving it in autologous cord plasma to prevent exposure to allogeneic or animal serum. This direct freezing of cord microparticles without previous culture expansion allows the processing and freezing of umbilical cord blood (UCB) and UC-MSCs from the same individual on the same day on arrival in the laboratory. UC-MSCs produce significant concentrations of hematopoietic growth factors in culture and augment hematopoietic colony formation when co-cultured with UCB mononuclear cells. Mice undergoing transplantation with limited numbers of human UCB cells or CD34(+) selected cells demonstrated augmented engraftment when UC-MSCs were co-transplanted. We also explored whether UC-MSCs could be further manipulated by transfection with plasmid-based vectors. Electroporation was used to introduce cDNA and mRNA constructs for GFP into the UC-MSCs. Transfection efficiency was 31% for cDNA and 90% for mRNA. These data show that UC-MSCs represent a reliable, easily accessible, noncontroversial source of MSCs. They can be prepared and cryopreserved under good manufacturing practices (GMP) conditions and are able to enhance human hematopoietic engraftment in SCID mice. Considering their cytokine production and their ability to be easily transfected with plasmid-based vectors, these cells should have broad applicability in human cell-based therapies.     

Immunotherapy of malignant melanoma in a SCID mouse model using the highly cytotoxic natural killer cell line NK-92.

In this work, we evaluated the potential of the natural killer (NK) cell line NK-92 and its IL-2-independent variants NK-92MI and CI, as immunotherapy for melanoma. In vitro, we found that NK-92 was much more cytotoxic to a number of human melanoma cell lines than lymphokine-activated killer (LAK) cells, particularly at low effector/target (E:T) ratios. In vivo treatment of mice challenged with MEWO melanoma cells with i.v. administered NK-92 and NK-92-MI resulted in a 1.5-2.5-fold increase in average length of survival. NK-92, MI, and CI were also effective against the WM1341 cell line, causing a 2-5-fold increase in survival when administered before the malignant cells. With s.c. injection, MEWO and WM1341 caused a primary tumor mass, secondary tumors, and metastatic cells. NK-92 cells reduced WM1341 primary tumor size by 40-90% and MEWO tumors by 30-75%. Similar results were seen with NK-92MI and CI. These data show that NK-92 cells are highly cytotoxic to human melanoma cells in vitro and in vivo and suggest that treatment with NK-92 cells may be a potentially effective immunotherapeutic modality in melanoma.     

Temporal Decrease of Trivalent Chromium Concentration in a Standardized Algal Culture Medium: Experimental Results and Implications for Toxicity Evaluation

The fate of two trivalent chromium salts (nitrate and chloride) in ISO algal culture medium was followed over 72 h; i.e., the typical duration of algal toxicity tests. Fifty percent of the initial Cr spikes was lost from the solutions by 24 h, with losses up to 90% after 72 h. Monitoring of the temporal variability of Cr(III) concentrations in algal culture media appears necessary to better characterize the toxicity of trivalent chromium to algae.     

Fibronectin restores defective in vitro proliferation of patients' lymphocytes after marrow grafting

In search for means of improving the impaired lymphocyte function of recipients after marrow grafting, we investigated the effect of fibronectin (FN) on patients' lymphocytes in allogeneic mixed lymphocyte cultures (MLC) and in cell-mediated lympholysis (CML) assays, since these tests are usually defective in transplanted patients. Four subgroups of marrow recipients were tested: patients within the first 100 days of transplantation (short-term) with (n = 16) or without (n = 14) acute graft-versus-host disease (GVHD), and long-term recipients with (n = 23) or without (n = 15) chronic GVHD. Exogenous FN (25 micrograms/ml) increased the proliferative response in the allogeneic mixed lymphocyte culture (MLC) significantly in cells from short-term patients without acute GVHD (+42%) and in those from long-term recipients with (+117%) and without chronic GVHD (+48%). In cells from patients with chronic GVHD, 3H-thymidine uptake after the addition of FN was enhanced to the level of that in lymphocytes of the corresponding marrow donor without exogenous FN. Fibronectin was effective only if added at the beginning of the MLC. In contrast to the results in MLC, exogenous FN failed to enhance phytohemagglutinin or OKT-3-induced lymphocyte proliferation and had no effect on CML activity. Moreover, FN did not show mitogenic activity in 3-6-day cultures. Our results demonstrate that FN in vitro is capable of restoring defective lymphocyte proliferation in marrow grafted patients, and circumstantial evidence suggests that this effect is mediated by an interaction between FN and monocytes.     

Bone marrow transplantation in patients aged 45 years and older

Increasing age has been reported to be a poor prognostic factor for survival after bone marrow transplantation. We evaluated causes of death and frequency and type of complications after marrow grafting in 24 syngeneic and 39 allogeneic recipients who were 45 to 68 years old at the time of transplant. Most patients were in an advanced stage of hematologic malignancy. Among patients given syngeneic transplants, actuarial disease-free survival at 7 years is 20%. The major causes of death were relapse of leukemia and idiopathic interstitial pneumonia. Among allogeneic recipients, 9 (23%) are currently alive, and actuarial disease-free survival at 7 years is 11%. Cytomegalovirus pneumonia and septicemia were the most frequent causes of death. Patients over 50 years of age had the poorest survival rate (1/13), but many of these were transplanted in an advanced stage of their disease. However, among 12 patients transplanted while in remission or at an early stage of their disease, 5 are surviving 65 to 1,160 days after transplantation, with an actuarial survival rate of 22% at 3 years. This is in contrast to those who received their transplant in relapse: 2 out of 20 patients (10%) became long-term survivors, with a probability of survival of 15% at 3 years. The actuarial incidence of grade II through IV acute graft- v-host disease (GVHD) was 30% for allogeneic recipients 45 to 50 years of age. This was not significantly different from the incidence in younger patients. In patients 51 to 62 years of age, the actuarial incidence of acute GVHD was 79%; however, this group included three partially HLA-mismatched transplants. Ten of 15 patients surviving at least 3 months developed chronic GVHD. These results suggest that marrow transplantation is feasible and should be considered in patients over 45 years, especially if recipients are in good clinical condition and are at an early stage of their disease, such as the chronic phase of chronic myelogenous leukemia and preleukemia. For patients more than 50 years of age, allogeneic marrow grafting cannot presently be considered first-line therapy.