Nonmyeloablative allogeneic stem cell transplantation for chronic lymphocytic leukaemia offers the possibility of disease control with minimal morbidity and mortality--a single institution experience.

Allogeneic stem cell transplantation is a treatment option for patients with poor risk CLL. We conducted a retrospective analysis of all CLL patients allografted at our institution, the University Hospital of Cologne, Germany. Data was collected on 40 patients from 2004 to 2012. The mean age was 54, and the majority were male (75 %). On average, the patients were diagnosed 6 years (range 2-12) prior to transplant with an average of 4 years (range 1-8) from time of first-line therapy to transplant. The remission states at the time of transplant were complete remission (CR) (n = 4), stable disease (n = 10), partial remission (n = 20) and progressive disease (n = 6). Only reduced intensity conditioning regimens were employed. The average CD34(+) cell dose was 4.16 × 10(6)/kg. Neutrophil engraftment was seen by day +17 (range 10-23) post-transplant, and 88 % achieved 95-100 % donor chimerism by day 100. Overall survival, progression-free survival and non-relapse mortality at 2 years post-transplant were 65, 52.5 and 27.5 %, respectively. A total of 51 % of patients were found to be minimal residual disease (MRD)-negative at 1 year post-transplant. Our single-centre experience confirms the valuable role of allogeneic stem cell transplantation (allo-SCT) in the treatment of poor risk CLL patients with promising long-term survival and acceptable transplant-related mortality. The advent of newer therapeutic agents should not hinder the consideration of allo-SCT for this patient cohort as it remains the only curative option for these patients.

[Allogeneic haematopoietic stem cell transplantation - an overview]. / Allogene Blutstammzelltransplantation - ein Überblick.

Allogeneic haematopoietic stem cell transplantation is an effective treatment option for chemotherapy-refractory or relapsed haematological malignancies such as leukaemias and lymphomas. After conditioning with chemotherapy with or without total body irradiation, donor cells are infused to reconstitute haematopoiesis. Donor-derived immune cells induce immune reactions to control or eradicate the underlying disease, thereby going beyond the effect of chemotherapy. This graft-versus-tumour effect (GvT) is often accompanied by detrimental graft-versus-host reactions (graft-versus-host disease, GvHD), which substantially influence the mortality and morbidity after transplantation. The balance between GvHD and GvT, implementing various parameters such as donor selection, stem cell source, conditioning, immune reconstitution and immunosuppressive regimens, represents the challenge in the field of allogeneic stem cell transplantation.

[Hemolytic anemia]. / Hämolytische Anämien.

Hemolytic anemia can be caused by various hereditary or acquired diseases. Classification is usually based on corpuscular or extracorpuscular defects. Beside the anemia, laboratory testing indicates increased lactate dehydrogenase, unconjugated bilirubin and reticulocytes as well as reduced or absent plasma haptoglobin. Knowledge of further diagnostic procedures (e.g., Coombs test, schistocytes, hemoglobin electrophoresis or flow cytometric analysis) leads in many cases to an underlying disease with differentiated therapeutic options. Autoimmune hemolytic anemia (AIHA) is often associated with diseases as HIV, connective tissue disease, lymphomas or malignant tumors and the hemolytic process is preexisting in many cases. Thrombotic microvascular diseases (e.g., thrombotic thrombocytopenic purpura or hemolytic-uremic syndrome) are further important causes of hemolytic anemia which need immediate diagnosis and treatment.

Two immunologically distinct human DNA polymerase alpha-primase subpopulations are involved in cellular DNA replication.

Metabolic labeling of primate cells revealed the existence of phosphorylated and hypophosphorylated DNA polymerase alpha-primase (Pol-Prim) populations that are distinguishable by monoclonal antibodies. Cell cycle studies showed that the hypophosphorylated form was found in a complex with PP2A and cyclin E-Cdk2 in G1, whereas the phosphorylated enzyme was associated with a cyclin A kinase in S and G2. Modification of Pol-Prim by PP2A and Cdks regulated the interaction with the simian virus 40 origin-binding protein large T antigen and thus initiation of DNA replication. Confocal microscopy demonstrated nuclear colocalization of hypophosphorylated Pol-Prim with MCM2 in S phase nuclei, but its presence preceded 5-bromo-2'-deoxyuridine (BrdU) incorporation. The phosphorylated replicase exclusively colocalized with the BrdU signal, but not with MCM2. Immunoprecipitation experiments proved that only hypophosphorylated Pol-Prim associated with MCM2. The data indicate that the hypophosphorylated enzyme initiates DNA replication at origins, and the phosphorylated form synthesizes the primers for the lagging strand of the replication fork.

Meibography and meibomian gland measurements in ocular graft-versus-host disease.

Meibomian gland loss in ocular GvHD was described as a mechanism contributing to dry eye and severe damage to the ocular surface. Infrared images of upper eyelid meibomian glands from 86 ocular GvHD patients, from 10 patients after allogeneic stem cell transplantation (aSCT) without ocular GvHD, from 32 patients prior to aSCT and from 30 healthy controls were analyzed retrospectively and evaluated using two grading schemes. The upper meibomian gland area (uMGA) was calculated and set in relation to the total tarsal area of the lid. Results demonstrate that meibomian gland loss is significantly increased in patients with ocular GvHD as well as in patients prior to aSCT in comparison with controls (P between 0.05 and <0.001). Patients after aSCT without ocular GvHD had no significant difference in uMGA in comparison with controls. This study suggests that meibomian gland loss in GvHD patients is likely to be a multifactorial process that also occurs prior to aSCT, possibly due to underlying diseases and/or secondary to chemotherapy or irradiation. In addition, the question has to be addressed whether meibomian gland loss could serve as a predictor for the development of ocular GvHD. Overall, infrared meibography should be included in routine examination of patients undergoing aSCT and during follow-up.

Ultrastructural characterization of a mouse melanoma cell line, a mouse fibroblastic cell line, and hybrids between them.

This report describes a transmission electron microscopic study on hybrids between subhexaploid mouse melanoma cells and subtetraploid mouse fibroblasts. The melanoma cell line was heterogeneous in terms of pigment production, but all cells contained melanosomes, although in different stages of development. Characteristic features of the fibroblasts included the cytoskeleton, endocytic vesicles, and occurrence of dilated cisternae of granular endoplasmic reticulum. Despite the gene dosage in favour of the melanoma parent cell, the hybrids were devoid of melanosomes, and their phenotype was typically fibroblastic in character.

Intracellular and extracellular localization of fibronectin in primary cultures of aortic smooth muscle cells using immunoperoxidase cytochemistry in electron microscopy.

This report describes the intracellular and extracellular localization of fibronectin at the ultrastructural level in primary cultures of aortic smooth muscle cells. Fibronectin was present in all the cisternae of the rough endoplasmic reticulum except the perinuclear cisterna, in large vesicles associated with the trans side of the Golgi complex, and in single large vesicles in the cytoplasm often associated with microtubules. The extracellular microfibrils were heavily stained. In sections parallel to the plane of growth bundles of extracellular microfibrils in continuity with arrays of intracellular microfilaments were observed (fibronexus). The basement membrane around the aortic smooth muscle cells was discontinuous and diffusely stained. The results indicate that fibronectin is localized in the cytoplasmic membranous apparatus of protein synthesis, processing, and secretion. The lack of reaction product in the flat cisternae of the Golgi complex let suggest either that fibronection may not be present in significant amounts within the flattened cisternae or that the method is insufficient in detecting the glycoprotein in this subcompartment off the Golgi complex.

The importance of the subendothelial connective tissue to the permeability of the neointimal barrier.

The thoracic aortas from two rabbits that had survived a single embolectomy catheter lesion for 2 years were studied by transmission electron microscopy after vital staining with Evans blue. An intimal thickening was formed inside the original internal elastic lamella in the re-endothelialized areas. Small blue areas in the ventral aortic wall showed intact endothelial cells covering an amorphous structureless subendothelial matrix. These observations indicate that endothelial cells do not form an effective barrier in the absence of differentiated subendothelial connective tissue.

Preservation of rabbit kidneys using a solution containing hydrolyzed starch.

An organ preservation solution has been developed by combining some features of the hypertonic citrate formulation of Ross, Marshall, and Escott (RME) with some features of UW solution. Specifically the solution (HP16) contains a balance of cations similar to that in RME and the same concentration of citrate, but sulfate is replaced by chloride and mannitol by a starch hydrolysis product (SHP). A gelatin-derived polypeptide (Haemaccel) is included to provide colloid osmotic pressure. The objective was to increase the effectiveness of RME by using a higher-molecular-weight osmoticum than mannitol, but avoiding the expense of raffinose; reducing the osmolality to a more physiological level; and including a colloid to make the solution suitable for continuous perfusion. The effectiveness of the solution was tested by 48-hr hypothermic preservation of rabbit kidneys. The results were compared with those obtained using RME or UW. It was shown that simple hypothermic storage was more effective than continuous perfusion, and that HP16 was more effective than RME and as effective as UW. The improvement over RME was ascribed to the isotonic osmolality and the inclusion of a higher-molecular-weight osmoticum (the SHP), possibly supplemented by the colloid (Haemaccel). Two SHP preparations, both with dextrose-equivalent values of approximately 35, were equally effective. These materials contain a standardized mixture of dextrose, maltose, and tri- and oligosaccharides, and have the osmotic properties of a trisaccharide. The results provide a new, inexpensive preservation solution that is as effective as any so far tested with this model, and they support the importance of appropriate osmotic properties for solutions to be used in organ preservation.

The effect on pregnancy of treatment with antibodies against pregnancy-associated murine protein I (PAMP-I) in inbred and outbred mice.

Intravenous injection of small amounts of monospecific rabbit IgG against pregnancy-associated murine protein I (PAMP-I) induced abortion in mice where there was a histocompatibility difference between mother and fetuses. No abortion could be induced in inbred mice by a similar treatment. The maternal serum level was found to be higher in inbred than in outbred mice. The abortive dose of antibodies did not influence the serum levels of PAMP-I. Histological examination of uterine, placental and liver tissue showed only morphological changes in the placental tissue of mice which aborted due to the treatment with anti-PAMP-I antibodies.

Immunohistochemical demonstration of pregnancy-associated plasma protein A (PAPP-A) in the syncytiotrophoblast of the normal placenta at different gestational ages.

The immunoperoxidase technique was used to study the localization of pregnancy-associated plasma protein A (PAPP-A) in formaldehyde-fixed paraffin-embedded tissue from normal human placentae at the gestational age of 8, 15 and 40 weeks. Sections of formaldehyde-fixed tissue treated with a proteolytic enzyme and incubated in antiserum against PAPP-A either raised in goats or rabbits showed that PAPP-A was distributed in the cytoplasm of the syncytiotrophoblast. The protein was not found in the cytotrophoblast. Sections without pretreatment with trypsin and incubation in goat anti-PAPP-A showed no staining reaction, whereas incubation in rabbit anti-PAPP-A revealed a staining of the syncytiotrophoblast surface. The results indicate that PAPP-A is probably synthesized in the syncytiotrophoblast.

The localization of pregnancy proteins (hPL, SP1 and PAPP-A) in intra- and extrauterine pregnancies.

The localization of human placental lactogen (hPL), pregnancy-specific beta-1 glycoprotein (Schwangerschaftsprotein 1, SP1) and pregnancy-associated protein A (PAPP-A) was examined in intrauterine and tubal ectopic gestation (n = 5) by the immunoperoxidase technique. The distribution of hPL and SP1 was identical in placental tissues obtained from intra- and extrauterine pregnancies, being uniformly seen throughout the syncytiotrophoblast. hPL and SP1 were not demonstrated in uterine decidual tissue from ectopic pregnancies. During early (week 8) intrauterine pregnancy, PAPP-A was not restricted to the mature syncytiotrophoblast, being observed also in some trophoblast-like cells adjacent to islands of syncytiotrophoblast. In contrast, in ectopic gestation, PAPP-A was observed in these cells at six weeks' gestation only. We were unable to detect PAPP-A in trophoblastic tissue of chorionic villi and uterine decidual tissue from ectopic gestation.

Using at least 5x10(6)/kg CD34+ cells for autologous stem cell transplantation significantly reduces febrile complications and use of antibiotics after transplantation.

For autologous stem cell transplantation, it is common practice to infuse at least 2 x 10(6)/kg CD34+ cells to ensure rapid engraftment. However it was recently claimed that increasing the threshold to 5 x 10(6)/kg leads to a faster platelet engraftment. To evaluate these threshold values in our patient population we undertook a retrospective analysis of 127 autologous transplants performed at our institution between 1992 and 1998. Diagnoses included Hodgkin's and non-Hodgkin's lymphoma, myeloma, acute leukaemias and solid tumours. The transplant was peripheral blood stem cells in 107 cases and CD34-selected peripheral blood stem cells in 20 cases. The median number of transplanted CD34+ cells was 3.2 x 10(6)/kg (range 0.64-25.9 x 10(6)/kg). Haematopoietic recovery to a neutrophil count >0.5 x 10(9)/l took a median of 10 (range 5-16) days from transplant. When comparing patients receiving at least 5 x 10(6)/kg and 2-5 x 10(6)/kg CD34+ cells we found a significant reduction in the median number of days with fever (1 vs 3.5 days, P = 0.0025), incidence of fever (78.8 vs 92.1%, P = 0.032) as well as duration of antibiotic treatment (7 vs 10 days, P = 0.038). This was paralleled by a faster neutrophil recovery to 0.5 x 10(9)/l (9 vs 10 days, P = 0.047). There was no significant difference in the number of platelet or red cell transfusions between the two groups. We conclude that transplantation with a stem cell dose of at least 5 x 10(6)/kg CD34+ cells reduces infectious complications and should thereby increase the safety of this type of therapy while reducing duration (and cost) of antibiotic therapy. The transplantation threshold should thus not remain at 2 x 10(6)/kg particularly in patients with a good stem cell mobilisation capacity.

Successful peripheral blood stem cell mobilization with etoposide (VP-16) in patients with relapsed or resistant lymphoma who failed cyclophosphamide mobilization.

High-dose chemotherapy (HDCT) followed by autologous blood stem cell transplantation is considered the treatment of choice for patients with relapsed or resistant aggressive non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD). However, several authors report failure of standard mobilization regimens in 29% to 56% of these patients making the completion of HDCT impossible and as a result, negatively influencing long-term outcome. Thus, effective new regimens for patients failing initial mobilization are needed. Here we report the results of using etoposide as a mobilizing agent in 16 patients with primary resistant or relapsed malignant lymphoma who had failed prior mobilization of peripheral blood stem cells (PBSC) with cyclophosphamide (4 g/m2) followed by G-CSF. The use of etoposide 500 mg/m2 (days 1-4) + G-CSF resulted in the successful collection of adequate numbers of PBSC with a median harvest of 3.6 x 10(6)/kg (range 2.2-12.6) CD34+ cells in all 16 patients. In 7/16 (44%) patients, the target yield of at least 2.0 x 10(6) CD34+ cells was harvested by a single apheresis and the maximum number of separations for all patients was two. No excessive toxicities appeared, allowing all patients to proceed to myeloablative chemotherapy. In addition, median peak values of circulating CD34+ cells were significantly higher after etoposide as compared to cyclophosphamide (49.2/microl vs 4.7/microl; P = 0.0004). These results indicate that etoposide + G-CSF is a highly effective mobilization regimen in patients who have failed cyclophosphamide mobilization.

Successful treatment of chronic disseminated candidiasis with caspofungin and itraconazole in a patient with progressive acute leukemia and prolonged neutropenia.

Severe fungal infections remain a significant cause of morbidity and mortality in neutropenic patients undergoing dose-intensive chemotherapy for malignant diseases. Chronic disseminated candidiasis (CDC) is a life-threatening complication in neutropenic patients because of the lack of responsive hematopoietic precursor cells. Resolution of Candida organ lesions after hematopoietic reconstitution may take months. Here, we report the case of a 19-year-old neutropenic woman with relapsed acute myelogenous leukemia and candidiasis of liver, spleen, and kidneys. Antifungal treatment was initiated using fluconazole and caspofungin but was changed to itraconazole and caspofungin. Despite elevated C-reactive protein (CRP) levels and detectable Candida organ lesions, antileukemic therapy was restarted with interleukin 2 at the same time as antimicrobial treatment. Eight weeks after the start of interleukin therapy, CRP levels and organ lesions were decreased significantly irrespective of continuing neutropenia. This case report describes the successful treatment of CDC during neutropenia using combination antifungal therapy and suggests controlled studies to establish optimal therapeutic strategies.

Atypical chronic myeloid leukaemia with trisomy 21 mosaicism as a sole chromosomal abnormality.

We present a 46-year-old patient with Ph-chromosome negative, bcr-negative chronic myeloid leukaemia (CML) in accelerated phase with a clonal trisomy 21 in the leukaemic blast cells. A rapid progress of disease with appearance of monocytosis is described, showing similar features to chronic myelomonocytic leukaemia (CMML). Heterogeneous characteristics and possible distinction of these two entities are discussed.

Fetal antigen 2 (FA2) in human fetal osteoblasts, cultured osteoblasts and osteogenic osteosarcoma cells.

Immunohistochemical staining techniques used on an 11-week-old fetus showed that fetal antigen 2 (FA2) was present intracellularly in endochondral and perichondral osteoblasts, and the immunoreaction was extended into the adjacent bone matrix. Osteoclasts and chondroblasts were found to be FA2 negative. A granular perinuclear intracytoplasmic FA2 immunoreaction was found in cultured osteoblasts and osteogenic osteosarcoma cells, and immunoelectron-microscopical examination revealed a granular immunoreaction product in the rough endoplasmic reticulum. These findings indicate that FA2 is synthesized by osteoblasts and osteogenic osteosarcoma cells. A reaction of immunological identity was found between FA2 purified from second trimester amniotic fluid and serum-free supernatants of cultured osteogenic osteosarcoma cells. This shows that an antigen recognized by the anti FA2 antibody is secreted by these malignant cells. Thus, FA2 may represent a marker for altered bone metabolism, and have a potential in the classification of osteogenic osteosarcoma/chondrosarcoma.

Fetal antigen 1 (FA1) in the human pancreas: cell type expression, topological and quantitative variations during development.

Monospecific rabbit anti-human fetal antigen 1 (FA1), was used to examine the distribution of FA1 during the development of the human fetal pancreas and liver using an indirect immunoperoxidase technique. FA1 was expressed by 94% of the glandular epithelial cells of the branching ducts in the pancreatic anlage at week 7 of gestation. This pattern changed during the development of the human pancreas, 64% of the glandular cells being FA1 positive at week 17 of gestation, decreasing to 11% in the infant (4 months after birth). In the infant and adults the FA1 expression was restricted to a subpopulation of beta-cells within the islets of Langerhans. Insulin immunoreactive cells were scattered throughout the epithelium of primitive branching pancreatic ducts at week 7 of gestation, well before the formation of islets. From the 7th through to the 17th week of gestation, FA1 was found in the cytoplasm of fetal hepatocytes, whereas no staining was observed in the liver from a 4-month-old infant. No FA1 expression was found in the epithelium of the developing gut. The present findings indicate that the glandular epithelial cells in the developing pancreas may serve as stem cells, which, if appropriately induced, may differentiate into endocrine cells. Fetal antigen 1 (FA1) may take part in or be a result of this differentiation.

Two fetal antigens (FA-1 and FA-2) and endometrial proteins (PP12 and PP14) isolated from amniotic fluid: localisation in the fetus and adult female genital tract.

Monospecific antisera against two fetal antigens (FA-1 and FA-2), alphafetoprotein (AFP) and two endometrial proteins (PP12 and PP14) were used to examine the distribution of these proteins and antigens in human trophoblast and gestational endometrium in first and third trimesters of pregnancy, normal human ovary and fetal tissues by indirect immunoperoxidase histochemical localisation techniques. Fetal liver stained exclusively for FA-1 and AFP which was used as a reference protein. Staining for FA-2 was seen in fetal connective tissue, in particular the basement membrane. FA-1 and FA-2 did not stain positively in decidua, trophoblast or ovarian tissue. Gestational endometrium stained positively for PP14 exclusively in the glandular epithelium, whilst staining for PP12 was seen only in the stromal cells. Trophoblast, both early and late, and ovarian tissue did not stain positively for any of the four substances tested.

Bicarbonate- versus lactate-based CAPD fluids: a biocompatibility study in rabbits.

Previous in vitro biocompatibility studies have shown bicarbonate-based continuous ambulatory peritoneal dialysis (CAPD) fluids to be superior to those based upon lactate/acetate. To evaluate these findings in vivo, 41 rabbits were subjected to CAPD for four weeks in a randomized prospective study using either Dianeal, a commercially available dialysis fluid containing lactate, or 87b, a bicarbonate-based CAPD fluid. Ten rabbits with CAPD catheters, which were flushed with a heparin solution every 36 hours, served as controls. None of the control rabbits showed clinical or histopathological signs of peritonitis, while 8 of 20 in the Dianeal group and 6 of 21 in the 87b group contracted peritonitis. Four rabbits in the Dianeal group had to be sacrificed early due to severe peritonitis. Post mortem examinations, including scanning and light microscopy, did not reveal any macroscopic or microscopic differences among the three groups of noninfected animals. No significant distinctions between the groups could be made for body temperature, weight gain, dialysate volume, dialysate differential leukocyte count, dialysate protein content, and food intake during the course of the study. In conclusion, the present animal model did not reveal any major difference in the biocompatibility between the lactate- and the bicarbonate-based CAPD fluids.