Large granular lymphocyte leukemia: An indolent clonal proliferative disease associated with an array of various immunologic disorders.

Large granular lymphocyte leukemia (LGLL) is a chronic lymphoproliferative disorder characterized by the proliferation of T or NK cytotoxic cells in the peripheral blood, the spleen and the bone marrow. Neutropenia leading to recurrent infections represents the main manifestation of LGLL. One specificity of LGLL is its frequent association with auto-immune disorders, among them first and foremost rheumatoid arthritis, and other hematologic diseases, including pure red cell aplasia and bone marrow failure. The large spectrum of manifestations and the classical indolent course contribute to the diagnosis difficulties and the frequency of underdiagnosed cases. Of importance, the dysimmune manifestations disappear with the treatment of LGLL as the blood cell counts normalize, giving a strong argument for a pathological link between the two entities. The therapeutic challenge results from the high rate of relapses following the first line of immunosuppressive drugs. New targeted agents, some of which are currently approved in autoimmune diseases, appear to be relevant therapeutic strategies to treat LGLL, by targeting key activated pathways involved in the pathogenesis of the disease, including JAK-STAT signaling.

[A microcytic sideroblastic anemia successfully treated with B6 vitamin]. / Une anémie microcytaire sidéroblastique carentielle traitée efficacement par de la vitamine B6.

INTRODUCTION: Sideroblastic anemia is a rare cause of microcytic anemia, which is characterized by ring sideroblasts on bone marrow aspirate. This anemia can be congenital or acquired. CASE REPORT: We report the case of an alcoholic 49-year-old man who presented with a severe microcytic sideroblastic anemia related to pyridoxine (B6 vitamin) deficiency. Acid folic deficiency was associated. The blood count normalized within one month after vitamin supplementation. CONCLUSION: Pyridoxine deficiency must be sought in sideroblastic anemia in patients at risk.

The Y chromosome effect on intermale aggression in mice depends on the maternal environment.

Two parental strains of laboratory mice, NZB and CBA/H, were chosen for their differences in attack behavior. NZB have higher scores than CBA/H. An effect of the Y chromosome on attack behavior was determined for two maternal environments. Each male was tested once in a dyadic encounter with an A/J male as a standard opponent. The two reciprocal F1s and the four reciprocal backcrosses were used. In each group, the proportion of attacking males was used as the dependent variable. In the first experiment, the ovarian graft method was used to test for an effect of variation of the overall maternal environment: parental vs. F1. The results demonstrated an interaction between the Y chromosome and the maternal environment. By use of the adoption method, it was shown in the second experiment that this maternal effect was probably postnatal (and not prenatal).

Further aspects of muscular dystrophy in mdx mice.

The histopathological aspects of leg muscles in young, adult and old mdx mice were compared. Before 1 yr of age, the mutant showed a vigorous regeneration, following the fibre necrosis, which led to hypertrophic muscles. Hypertrophy resulted from a mixture of hypertrophic and small fibres but not from fibrosis. From the age of 15 months, the muscles progressively atrophied and the fibres were atrophic or split. An important degree of fibrosis occurred particularly in soleus and plantaris muscles. These aspects in old mdx mice are similar to those described in other dystrophinopathies in animals and humans.

Transplantation of adult-derived myoblasts in mice following gene transfer.

We have explored the use of myoblasts obtained from adult animals as a target for somatic gene therapy. Myoblasts from an adult beta-glucuronidase deficient (MPS VII) mouse were isolated and infected with a retroviral vector carrying the human beta-glucuronidase cDNA. Beta-glucuronidase was used as a reporter gene to follow the fate of genetically-modified myoblasts after transplantation into the tibialis anterior of MPS VII recipients. When experimental necrosis had been induced in the recipient muscle prior to cell injection, histological analysis demonstrated efficient engraftment of adult derived myoblasts following gene transfer. The reconstituted myofibres expressed the transgene for at least 10 weeks following transplantation.

Morphological and functional study of extensor digitorum longus muscle regeneration after iterative crush lesions in mdx mouse.

The regenerative capacity of mdx Extensor Digitorum Longus (EDL) muscle after iterative muscle crush injuries was examined and compared with that of age-matched control C57BL/10 mice. Muscle crush injuries were performed at 8 weeks and repeated at 12 and 16 weeks. Contralateral non-crushed EDLs from mdx and C57BL/10 mice were used as internal controls for histopathology, histoenzymology, morphometry and for the study of the contractile properties. Morphological examinations were performed at 12, 16 and 20 weeks, respectively one month after a single, a second or a third crush. Contractile properties were studied at 12 to 20 weeks. By 20 weeks, no difference in the number of fibres with internal nuclei could be observed between crushed EDL from both strains, and non-crushed mdx EDL; the area and the diameter of crushed EDL from mdx mice were, respectively, 1.5- and 1.2-fold higher than the ones from crushed EDL from C57BL/10 strain. By 20 weeks, diameter distribution of crushed EDL muscles from C57BL/10 mice were shifted towards smaller fibre diameter, whereas in mdx mice, diameter distribution of crushed EDL muscles paralleled that of non-crushed EDL muscles. By 20 weeks, crushed mdx and C57BL/10 EDL muscles produced 77 and 47% of normalized tetanus tension respectively of non-crushed mdx and C57BL/10 EDL muscles. Following crush injury, both 12- and 20-week mdx and C57BL/10 EDL exhibited a slowed time to peak (TTP) and half-relaxation time (H1/2R) of twitch. There was no difference in posttetanic potentiation between the different groups. Crushed EDL of both strains showed an increased resistance to fatigue, compared to the non-crushed controls. The present study provides morphological and functional evidence for the greater recovery of mdx muscle compared to C57BL/10 muscle following iterative crush injury; however, the recovery does not completely prevent the appearance of necrosis/regeneration features.

Expression of NCAM and its polysialylated isoforms during mdx mouse muscle regeneration and in vitro myogenesis.

In order to understand the mechanism of the muscular regenerative process which occurs in mdx mice, the expression of neural cell adhesion molecule (NCAM) isoforms and their polysialylated (PSA) derivatives were studied during the postnatal development of normal and mdx mice in relation to the stage of the regeneration of muscle fibres, in the quadriceps. NCAM expression was also examined during in vitro differentiation of satellite cells isolated from both mdx and normal muscles. The immunohistochemical and biochemical analyses were done using antibodies for the different isoforms. The data presented here suggest that before the onset of necrosis and regeneration, the expression of NCAM isoforms in the quadriceps of mdx mice was similar to normal mice. Later, NCAM and PSA-NCAM expression in mdx mice increased and was related to the muscular regenerative process, and the overall level of NCAM expression can be considered as a good index of muscle regeneration. Young regenerative fibres expressed NCAM and PSA-NCAM, while mature regenerative fibres, in which myonuclei remained centrally located, did not express either NCAM or the PSA isoforms. Therefore, in terms of NCAM expression, the fibres in mdx muscle with centrally located nuclei appeared similar to mature fibres found in normal adult muscle. A major form of 145 kDa and a minor form of 115 kDa were detected in mdx regenerative muscle. The 145 kDa NCAM was sialylated, as demonstrated by its sensibility to exoneuraminidase which generates a desialoform of 125 kDa, but not polysialylated since it was not recognized by the anti-MenB antibody, specific for PSA-NCAM. In contrast, the molecular forms of NCAM migrating as a broad band from 160 kDa to 220 kDa were identified as PSA-NCAM. The comparison of in vitro differentiation of normal and mdx satellite cells showed that the expression of NCAM isoforms by mdx cells was similar to that expressed by normal cells. Both our in vivo and in vitro data concerning NCAM expression show that regeneration in mdx mice does not differ from that observed in other necrotic diseases. In other words, NCAM is unlikely to be a dystrophin-associated molecule since lack of dystrophin does not affect its expression.

mdx mice show progressive weakness and muscle deterioration with age.

The time-course of degeneration/regeneration was investigated in leg muscles throughout the life of the mdx mutant mouse, which is a biochemical homologue of Duchenne muscular dystrophy (DMD). In young and adult mice (up to 52 weeks old), muscle fibre necrosis was compensated by a vigorous regeneration, but in old mdx mice (65-104 weeks) this regeneration slightly declined, while the necrotic process persisted. Body and muscles weights declined strikingly after 52 weeks. Life span of mdx mutants was reduced in comparison with the control C57BL/10 animals. Immunostaining of old mdx muscles showed clusters of dystrophin-positive fibres. Muscle fibres in old mdx mice showed great variation in size, many being atrophied or split. Endomysial fibrosis became increasingly conspicuous, and there was some accumulation of adipose tissue. These progressive degenerative changes of old mdx mice resemble those found in DMD and imply that basic pathological similarities between the murine and human diseases previously observed in diaphragm of mdx mice may be extended to other skeletal muscles.

Time course study of the isometric contractile properties of mdx mouse striated muscles.

The isometric twitch and tetanic contractions of three hindlimb muscles (soleus, plantaris, extensor digitorum longus) were recorded in situ in groups of mdx and C57BL/10 control mice at young, adult and old ages (3, 4, 6, 8, 13, 26, 39 and 52 weeks). Based on a two-way analysis of variance (age/phenotype) the mdx phenotype did not modify the absolute tension but was associated with a significant decrease in the tetanic tension normalized to muscle weight in all the muscles which became heavier. These results suggest that the contractile material in mdx is not so powerful as in controls. Moreover, significantly faster time to peak and half-relaxation time were observed in mdx soleus and plantaris. Comparison between these contraction characteristics and those of other experimental models suggests that the high percentage of regenerated fibres in mdx muscles could play a role in modifying contractile properties.

Fibres of intermediate type 1C and 2C are found continuously in mdx soleus muscle up to 52 weeks.

The mdx mutant is a murine homologous model of Duchenne muscular dystrophy (DMD). Fibre types determined by the myosin-ATPase technique in soleus muscles were compared in C57BL/10 control and mdx mice from 3 to 52 weeks of age. The control strain continuously presented 70% of type 2 fibres whereas the mdx strain showed an increase to 80% at 6 weeks and a subsequent decline. In mdx muscles, necrosis which begins at 3 weeks of age did not affect specifically one type of fibre. Type grouping was never observed when muscle regeneration occurred. Fibres of intermediate type (1C and 2C) were found continuously up to 52 weeks of age in the mdx mutant. The foci of small immature regenerating fibres were of type 2C but never 1C. A few mature fibres were either of type 2C or 1C. We suggest that the presence of intermediate type fibres could result from the co-expression of type 1 and 2 myosin heavy chains, indicating a transition from type 2 to type 1 in regenerating fibres.

Age-related differences in regeneration of dystrophic (mdx) and normal muscle in the mouse.

The mdx mouse, a genetic homologue of human Duchenne muscular dystrophy (DMD), has been attributed with a greater regenerative capacity of its skeletal muscles. Here, we have tested the hypothesis that muscles of mdx mice regenerate better than those of nondystrophic animals. We studied muscle regeneration resulting from a denervation-devascularization injury (DD) of extensor digitorum longus muscle (EDL) at 3 weeks and 2 months in mdx and wild-type (C57BL/10) mice. Histological and morphometrical studies of muscle regeneration were made from 3 to 180 days later. When DD was performed in 3-week-old C57BL/10 mice, the percentages of nonperipheral nuclei in regenerated fibers decreased progressively over 3 months. This decrease did not occur in animals where DDs were performed at 2 months, suggesting that two different populations of muscle precursor cells are mobilized in muscle regeneration in mice at these two ages. Moreover, mdx EDL muscle regenerated similarly to the controls for up to 60 postoperative days, as shown by distribution of mean diameters and percentage of nonperipheral nuclei of muscle fibers. After 60 postoperative days, necrosis/regeneration characteristics of mdx muscles recurred, suggesting that mdx-regenerated muscle fibers remain susceptible to degeneration.

Phenotype of dystrophinopathy in old mdx mice.

BACKGROUND: Mdx mutant mice, like patients with Duchenne Muscular Dystrophy (DMD), lack dystrophin, a subsarcolemmal protein, that results in myofiber necrosis. However young mdx mice, in contrast to DMD children, exhibit a successful muscle regeneration and not an extensive fibrosis. METHODS: Old mdx mice were monitored clinically up to their spontaneous death, and most of their organs were studied histologically to look for differences with those of the wild C57BL/10 mice strain. RESULTS: In old mdx mice (at least 20 months of age), we report clinical and pathological features of muscular dystrophy, i.e., progressive motor weakness and loss of myofibers replaced by extensive connective tissue, similar to the phenotype of dystrophinopathy observed in DMD patients. Various degrees of dystrophic involvement were observed in cardiac, respiratory, postural, and hindlimb skeletal mdx muscles and also in smooth muscles of the digestive and urinary tracts. No gross histological abnormalities were found in other tissue than muscular tissue. CONCLUSIONS: Late in life, mdx mice develop a muscular dystrophy close to DMD dystrophinopathy. We suggest that the study of the effects of ageing in mdx mice would give clues to better understand the pathophysiology of DMD.

[Frequency of platelet alloantigens in the Spanish population]. / Frecuencia de los aloantígenos de las plaquetas en población española.

PURPOSE: The aim of this study is to investigate the distribution and incidence of platelet alloantigens in Spain and to compare this frequency with that found in other populations. MATERIAL AND METHODS: Five hundred blood donors were phenotyped for the HPA-1a (Zwa), HPA-3a (Bak(a)), HPA-4a (Yukb/Pen(a)) y HPA-4b (Yuk(a)). One hundred of these donors were also phenotyped for the HPA-5a (Br(a)) antigen. Furthermore, we also phenotyped a group of 50 black individuals from a community of west African immigrants who live in the province of Barcelona. The typing of all the antigens except those of the HPA-5 system was carried out using the technique of Indirect Immunofluorescence. To type the antigens of the HPA-5 system the MAIPA technique was employed. RESULTS AND CONCLUSIONS: The incidence of the different platelet specific antigens in the Spanish population is, in general, the same as that detected in the other European countries and in the white population of the USA. In Europe and North America, the HPA-1a antigen is the most important of all owing to its involvement in alloimmune clinical disorders. In Asia, this role seems to be played by the HPA-4b antigen. Moreover, polymorphism for the HPA-1a allele does not seem to exist. The incidence of the HPA-5b (Br(a)) antigen in Spain, which is higher than that for France and Germany, suggests a high involvement of this antigen in our country in different alloimmune processes. The preliminary data obtained in a group of black west Africans suggest the absence on polymorphism for the HPA-1a allele and that the incidence of the remaining antigens is comparable to that of the Spanish population.

T-cell-dependent fibrosis in the mdx dystrophic mouse.

In Duchenne muscular dystrophy patients, the pathological hallmark of the disease, namely, the chronic accumulation of sclerotic scar tissue in the interstitial space of skeletal muscle is attributed to manifestation of secondary pathological processes. Such anomalous generation of matrix protein is thought to be driven by the continuous degeneration and regeneration of muscle both in Duchenne Muscular Dystrophy and in the mdx mouse homolog. We examined mdx and the control strain C57bl/10 mice over a range of ages with respect to the amounts of collagen present in muscles and other organs, finding that the mdx have significantly higher collagen content at later time points in their kidney and lung as well as their muscles. Surprisingly, when we bred the mdx mice on the nu/nu background, the time course of fibrogenesis was modified depending on the tissue and the collagen content was significantly different in age-matched mice. Transplantation of normal thymic tissue into the mdx-nu/nu mice replenished their T-cells and concomitantly altered the collagen content in their tissues to levels comparable with those in immunocompetent mdx mice. This suggests that T-cells play a role in the onset of the fibrotic events that undermines the ability of dystrophic muscle to regenerate.

Computerised dystrophic muscle simulator: prospecting potential therapeutic strategies for muscle dystrophies using a virtual experimental model.

Inherited muscle diseases are often characterised by widespread muscle damage in the body, limiting the clinical relevance of cell or gene therapy based upon direct injections into muscles. Recent studies have shown, however, that cells originating from the bone marrow are able to target necrosis-regeneration sites as they occur and, in addition, may also participate in the muscle regeneration after undergoing myogenic differentiation. Here, we present a computerised dystrophic muscle simulator that allows the prospecting of different scenarios of both disease evolution and appropriate employment of blood-borne cells as therapeutic shuttles. It provides the option of examining their use either to transfer a healthy gene into the tissue or to impart substances designed to boost its regeneration. One of the major advantages of this tool is that it offers the opportunity of visualising and composing therapeutic strategies in virtual paradigms in which severe clinical situations, not necessarily available in animal models, can be created. The dystrophic muscle simulator is freely accessible via the Genethon web site (www.genethon.fr), and in the online version via http:@www.wiley.co.uk/genmed.

[Non-hemolytic transfusion reactions of the febrile type]. / Reacciones transfusionales no hemolíticas de tipo febril (RTNHF). Estudio serológico de 100 casos.

The results of a hundred cases of febrile non haemolytic transfusion reactions are presented. We used the techniques that would best identify the different causes of these reactions. Most of these antibodies were anti HLA. Platelet and granulocyte-specific antibodies were seldom detected. The nature of the antibodies was specified by their different reactivity with the cells of various donors, by applying a panel of cells from typed donors and by absorption experiments. The study of the donor sera enabled us to elucidate the causes of some reactions. Nevertheless, in a significant percentage of cases it was not possible to determine the cause of the reaction, especially in the case of reactions secondary to platelet transfusions. Finally, we analyzed the reasons that could account for these phenomena.

Characterization of antibodies directed against platelet cryptantigens detected during the immunological study of 356 consecutive patients with presumed autoimmune thrombocytopenia (AITP).

Cryptic antigens are detected by antibodies present in a wide spectrum of patients with or without thrombocytopenia, and even in healthy individuals. They are produced for unknown reasons and do not react with antigens of native platelets, but only with altered platelets. Cryptantigen antibodies may not only result in spuriously low platelet counts, but also in 'falsely' positive tests for platelet antibodies. We report our experience in the characterization of the different types of antibodies directed against cryptantigens of platelets: EDTA-dependent antibodies, PFA-dependent antibodies, EDTA-PFA-dependent antibodies and cold agglutinins. These antibodies were detected in the course of the serological study of 37 patients from a group of 356 (10%) whose blood was sent to our laboratory for platelet antibody testing. Pseudothrombocytopenia was diagnosed in 24 cases. Twenty-one of these showed EDTA-dependent or EDTA-PFA-dependent platelet agglutination and three were due to the presence of cold agglutinins. In 13 patients the thrombocytopenia was genuine. Eleven of these presented EDTA-dependent or EDTA-PFA-dependent antibodies in their serum and in the two remaining cases PFA-dependent antibodies were found. Cryptantigen antibodies were also detected in 9 out of 228 (4%) blood donors who were used as healthy controls in the platelet immunofluorescence test. In the light of the results obtained we put forward some guidelines to detect the presence of these antibodies and establish an accurate serological and clinical diagnosis of the autoimmune thrombocytopenias.