Biomedical subjects

J Lorenzen

Publications and source records attributed to J Lorenzen.

At least 73 records · Page 4Linked to original sources

PubMed1996

Apoptosis in acute myeloblastic leukemia: follow-up study on trephine biopsies of the bone marrow.

A clinicopathological study on 87 adult patients presenting with "de novo" acute myeloblastic leukemia (AML) was performed to assess the rate of apoptosis before and during chemotherapy and its predictive impact on clinical course. Evaluation included trephine biopsies of the bone marrow and the situ end-labeling technic (ISEL) for the identification of programmed cell death in large and intact hemopoietic tissue areas. In comparison with a control group of 21 patients without any hematological disorder, morphometric analysis revealed no significantly different numbers of apoptotic cells in AML at the onset of disease and following sequential examinations at intervals ranging between 10 to 19 months. Moreover, the incidence of programmed cell death was not associated with the subgroups of the FAB classification and statistics failed to show a relationship with survival or remission status. In conclusion, these findings are in keeping with the assumption that apoptosis occurs with the same frequency in recovering normal hemopoiesis in complete or partial remission, in manifest AML and relapse. In the latter conditions, enhancement of proliferation is not associated with an increase in the apoptotic index.

Acute Disease↗

PubMed1996

Nasal T/NK cell lymphoma: a clinico pathologic study of 30 west Chinese patients with special reference to proliferation and apoptosis.

Midfacial T-cell lymphomas are more prevalent in Asia than in Europe or North America. Clinically, these lymphomas are noted as one major differential diagnosis in the malignant midline granuloma syndrome. During the past years, the group of nasal T/NK cell lymphomas has been recognized that is frequently associated with EBV-infection. The aim of the current publication was to describe the clinical presentation and course of 30 patients attending the West China University of Medical Sciences, Chengdu, P.R. China, between 1991 and 1994. Clinical records were assessed and the patients were followed for 6 to 29 (mean 12.4) months. Several microscopic features thought to be associated with this entity were carefully evaluated together with immunohistochemical data. The proliferation of the tumour cells was assessed by determining the mitotic index and the ratio of MIB-1 labelled cells. In addition, the incidence of apoptotic cells was investigated by means of the in-situ end labelling (ISEL) technique. Our data confirm the expression of T-cell markers by T/NK cell lymphomas as determined by the immunohistochemistry. The apoptotic index was found to correlate with the ratio of MIB-1 labelled cells. Expression of the bcl-2 oncoprotein was not associated with increased or diminished proliferation or cell death, respectively. Eight of the thirty patients succumbed to their disease during the follow-up period. Kaplan-Meier cumulative survivals and log-rank tests revealed a significant impact of MIB-1 labelling on mean survival times.

Adolescent↗

PubMed1996

[Individual therapy goals: on outcome documentation of inpatient psychotherapy from the patient and therapist viewpoint].

The results of a study evaluation in practicability of two newly developed documentation forms are presented. Examined are 82 inpatient treatment episodes in an already published clinical concept with different treatment settings. Parallel versions of the forms were completed by patients and therapists. The 'Erge-Doku-A-Form' allows for the naming of up to five therapy goals determined at the beginning of therapy and evaluated in relation to their achieved quality at the end of therapy. The 'Erge-Doku-B-Form' describes a variety of problem areas as well as questions related to medication and changes induced by therapy. Surprising there were a high number of 230 Individual Therapy Goals (ITG) by patients and 262 ITG by therapists which could be arranged into 89 content categories and 5 main categories. Outcome measurement shows different results. There was a significant relationship between the 'well-being', the impression of 'satisfying treatment' at the end of the inpatient period and 'success in the main ITG'. The documentation forms presented here allow an outcome-measurement depending on differential indications.

Adult↗

PubMed1996

AIDS-related bone marrow lesions--myelodysplastic features or predominant inflammatory-reactive changes (HIV-myelopathy)? A comparative morphometric study by immunohistochemistry with special emphasis on apoptosis and PCNA-labeling.

There seems to be general agreement that AIDS-related bone marrow changes are consistent with myelodysplastic features. To re-evaluate this assumption more critically, a comparative study was performed on marrow smears and trephine biopsies in 20 patients presenting manifest stages of AIDS and in 30 patients with primary myelodysplastic syndromes (MDS). For an assessment of cytological atypias enzyme-(naphthol AS-D-chloroacetatesterase) and immuno-histochemical techniques including monoclonal antibodies directed against erythro-(Ret40f) and megakaryopoiesis (CD61), macrophages (PG-M1) and proliferating cell nuclear antigen-PCNA (PC10) were applied and staining results determined by morphometric analysis. The in situ end-labeling (ISEL) technique was used for the demonstration of cells undergoing apoptosis (programmed cell death). In the HIV-infected bone marrow the frequency of erythroid precursors and macrophages exceeded significantly the corresponding counts in MDS. In comparison with a control group, megakaryocytes were increased in both entities under study. However, MDS was characterized by a prevalence of atypical microforms. The small-sized megakaryocytes in MDS revealed striking abnormalities of nuclear-cytoplasmic organization. Severe defects of nuclear lobulation (pseudo-Pelger anomalies) of mature neutrophils or nuclear bridging of erythro-normoblasts were not conspicuous in AIDS. Whereas the incidence of apoptosis was significantly higher in MDS, no such differences could be found comparing the PCNA-labeling index of AIDS and MDS. On the other hand, both parameters were significantly increased in comparison with the controls. In the HIV-infected bone marrow alterations of the myeloid stroma were most prominently expressed. If well defined criteria for the diagnosis of (myelo-) dysplasia were applied, our findings in AIDS were not in keeping with the assumption of significant maturation defects comparable with MDS. Hence, bone marrow lessions accompanying AIDS should be separated from MDS and should be diagnosed as HIV-myelopathy.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Effects of interferon treatment on the macrophage population in the bone marrow of patients with Ph1+-CML.

In Ph1+-CML the abnormal function of bone marrow stroma was related to the presence of clonally transformed macrophages (MAs). Moreover, previous in vitro studies revealed that activation (phagocytosis, cytotoxicity) of MAs was associated with a pronounced increase in alpha-D-galactosyl residues on their membranes. Stimulation of this cell population has been shown to be easily accomplished by interferon (IFN) treatment. The latter caused an enhanced expression of binding sites for the lectin Griffonia simplicifolia isotype I-B4 (GSA-I), specific for this carbohydrate moiety. The present immuno- and lectinhistochemical study was designed to quantify MA subsets of the bone marrow in patients with Ph1+-CML under IFN therapy. For comparison a control group with monotherapy by busulfan (BU) was included. Identification of the total MA population was carried out by a monoclonal antibody against CD68 (PG-M1) and for the characterization of its activated fraction, the lectin GSA-I was employed. In both therapeutic groups morphometric analysis revealed a conspicuous increase in PG-M1-positive MAs in sequential trephine biopsies. However, following IFN therapy the relative amount of the GSA-I fraction was maintained or even increased and accompanied by enhanced apoptosis. On the other hand, BU generated a significant reduction of this subpopulation and the number of apoptotic cells as well. This finding is probably related to the immunomodulatory activity of IFN associated with MA activation and secretion of biogenic mediators. These are thought to belong partly to the so-called tumor necrosis factor superfamily, which is known to stimulate programmed cell death (apoptosis).

Adolescent↗

PubMed1996

[A comparative study of apoptosis and proliferation of nasal T cell lymphoma].

A quantitative evaluation and statistical analysis of apoptosis and proliferation in 30 cases of MMR was carried out by means of in situ end-labelling (ISEL) of fragmented DNA technic and expression of Ki-67 antigen in routine paraffin sections with comparison of clinicopathology and prognosis. 28 patients had been followed for 6 to 29 months (mean 12.4 months) and 8 (28.5%) died of relapse and idsease progression. The results showed that (1) the number of apoptotic cells was found to correlate with the ratio of Ki-67 positive cells (P < 0.0063). No significant association of mitotic figures, Ki-67 labelled cells and the expression of bel-2 oncoprotein was observed. The results also did not suggest a correlation of proliferation or apoptosis with the stage of the disease at presentation. (2) statistical analysis revealed a significant impact of Ki-67 positive cells (P < 0.0160) on mean survival time and no significant association among apoptosis, morphologic changes and the mean survival time was found in this group of cases. The relation between apoptosis, proliferation, expression of bcl-2 oncoprotein and EBV infection in related lymphomas were discussed.

Adult↗

PubMed1995

[Brain and gallbladder metastases 10 years after local excision of a cutaneous malignant melanoma].

A 52-year-old man had a generalized seizure resulting in fracture of two vertebrae 10 years after excision of a superficially spreading melanoma (Clark level III) on the left flank. Additional to pain on pressure over the right upper abdomen, the erythrocyte sedimentation rate was increased (41 mm) and the activity of the liver enzymes was raised. Neurological examination indicated a loss of short-term memory, signs of meningeal irritation, unsteady gait and falling tendency. Cerebrospinal fluid showed mild pleocytosis and cells suspicious of tumour. Upper abdominal ultrasonography revealed an enlarged gallbladder with contents of soft-tissue density. Magnetic resonance imaging showed numerous round lesions in the brain which looked nodular after contrast-medium injection. Cholecystectomy revealed metastases of a malignant melanoma. It was likely that the cerebral lesions also represented metastases. The confusional state increased in severity. Neither cytostatic drugs nor radiotherapy were administered, because of the poor prognosis. The patient, now fully disoriented, died 54 days after hospital admission.

Brain Neoplasms↗

PubMed1995

Secretion of cytokines (interleukins-1 alpha, -3, and -6 and granulocyte-macrophage colony-stimulating factor) by normal human bone marrow megakaryocytes.

The effects of cytokine stimulation [recombinant human interleukin (rhIL)-1 alpha, rhIL-3, rhIL-6, rhIL-11, and rh granulocyte-macrophage colony-stimulating factor (GM-CSF)] on the secretory activity of normal human megakaryocytes were studied by means of the reverse hemolytic plaque assay (RHPA) in enriched cell preparations. This test facilitates an extremely sensitive determination of cytokine secretion at the single-cell level, together with the clear-cut identification of each immunostained (CD61) secretory active megakaryocyte. Moreover, the reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the expression of IL-6, IL-6 receptor (IL-6R), IL-9, IL-10, IL-12, and IL-13 mRNA in highly concentrated megakaryocyte preparations. In comparison with the spontaneous secretion rate, stimulation with rhIL-1 alpha, rhIL-6, and rhGM-CSF failed to induce a significant increase in the release of cytokines by CD61+ cells. On the other hand, both rhIL-3 and, in a less pronounced way, rhIL-11 exerted a marked effect on IL-6 secretion. Additionally, after stimulation with rhIL-3, a significant enhancement of the secretion of IL-3 and GM-CSF, but not of IL-1 alpha, could be observed. Using the RT-PCR, a significant induction of IL-6 expression could be appreciated in the enriched megakaryocyte population (60% to 80%) stimulated with rhIL-3. The results of this study provide persuasive evidence that a number of cytokines are synthesized and secreted by human megakaryocytes and not only by hematopoietic stroma cells. These data suggest the existence of autocrine and paracrine mechanisms that may influence maturation and differentiation of megakaryocytes as well as act on various stroma cells to sustain an appropriate hematopoietic micro-environment.

Base Sequence↗

PubMed1995

[Apoptosis in Hodgkin's disease].

Previous studies have concentrated on the proliferative behaviour of the neoplastic cell compartment in Hodgkin's disease (HD). The aim of the current investigation was to analyse the frequency of programmed cell deaths in Hodgkin and Reed-Sternberg (HRS) cells in the different subtypes of HD and to correlate this phenomenon with the expression of the bcl-2 oncogene. For this purpose, we investigated paraffin-embedded material from 63 cases of HD. Oncogene expression was determined by immunohistochemistry with the monoclonal antibody bcl-2-124. The detection of apoptotic cells was facilitated by application of the in situ end-labelling (ISEL) technique. Our results confirmed that bcl-2 expression is low in the lymphocyte-predominant subtype of HD. Apoptotic cells were found in all subtypes to a variable extent and were not significantly associated with any particular subtype. Interestingly, there was no correlation of bcl-2 expression and the presence or absence of apoptotic HRS cells. Hence, other factors must be operative in the regulation of programmed cell death in HD. Such mechanisms have been described for lymphocytes under various conditions, such as negative selection in germinal centres and within the thymus, DNA damage due to irradiation, and cellular cytotoxicity.

Apoptosis↗

PubMed1995

[Extraneural metastasis of brain and spinal cord tumors. Report of 2 cases].

This case report refers to two patients with the rare entity of an extraneural metastasizing central nervous system tumor. The first patient presented with ipsilateral cervical lymph node metastases 4 years after a diagnostic biopsy and 2 years after removal of an anaplastic oligodendroglioma, respectively. The origin of the metastases was confirmed by immunohistochemistry. The second case described was found in a 26-year-old female who had suffered from a spinal cord pilocytic astrocytoma in infancy and been treated by surgery and radiotherapy. On postmortem examination, transformation to a primitive neuroectodermal tumor was found. Morphological and immunohistochemical features of the metastases were exactly identical with those of the primary tumors in both cases. The pathomechanisms of metastasizing CNS tumors are discussed with reference to the reasons why such cases are so rarely observed. The incidence of remote metastases and differences depending on tumor type are estimated. No relation to malignancy grade can be detected.

Adolescent↗

PubMed1995

Hodgkin cells accumulate mRNA for bcl-2.

BACKGROUND: The bcl-2 oncogene is able to prevent cells from apoptosis. Overexpression of the bcl-2 protein seems to be important for the pathogenesis of follicular center cell lymphomas, in which both protein and mRNA usually show high levels. In addition, the expression of the Epstein-Barr virus-encoded late membrane protein up-regulates the bcl-2 protein in cell lines. The aim of the current study was to investigate the expression of the bcl-2 oncogene in Hodgkin's disease both at the protein and mRNA level in correlation with the expression of the late membrane protein. EXPERIMENTAL DESIGN: Thirteen cases of all histologic types of Hodgkin's disease, six cases of chronic nonspecific lymphadenitis, three tonsils with follicular hyperplasia, seven cases of follicular small cleaved cell lymphoma, and six cases of follicular large cell lymphoma, were analyzed. We designed a novel digoxigenin-labeled oligonucleotide probe complementary to bcl-2 mRNA for nonisotopic in situ hybridization. Bcl-2 oncoprotein and late membrane protein expression were determined by immunohistochemistry. The presence of the 14;18 translocation was analyzed by PCR for the major breakpoint region. RESULTS: The main finding was that, irrespective of subtype, the vast majority of Hodgkin cells express abundant bcl-2 mRNA. Oncoprotein expression, however, varied from case to case, with the highest prevalence in the nodular sclerosing subtype, and showed no strict correlation with the late membrane protein. In our case, no 14;18 translocation could be found in Hodgkin's disease. CONCLUSIONS: Hodgkin cells in all types of Hodgkin's disease demonstrated high levels of bcl-2 mRNA, while the bcl-2 protein expression was inhomogenous. In nodular lymphocyte predominant type, the bcl-2 mRNA and protein pattern is comparable to germinal center cells. This finding is a further argument for the germinal center cell origin of this type of Hodgkin's disease.

Base Sequence↗

PubMed1995

Detection and quantification of transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF) release by normal human megakaryocytes.

Transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF) are known as protein cytokines involved in differentiation as well as in maturation processes within the hematopoietic system. Both enhance the proliferation and/or collagen synthesis in fibroblasts and are found within the alpha-granules of megakaryocytes. To learn more about the regulation mechanisms involving synthesis and secretion of these cytokines it is important to develop suitable experimental conditions. We have applied the reverse hemolytic plaque assay (RHPA) to CD61+ megakaryocytes prepared from bone marrow of hematologically normal patients. By means of the RHPA, the spontaneous and stimulated secretion of TGF-beta 1 and PDGF could be analyzed at the single cell level. According to morphometric analysis, predominantly small megakaryocytes including precursors (pro- and megakaryoblasts) secrete TGF-beta 1 and PDGF under physiological conditions. Furthermore, the proportion of actively secreting megakaryocytes increased significantly following treatment with recombinant human (rh) IL-3 for 8 h. A slight induction was also appreciated after stimulation with interleukins rhIL-1 or rhIL-11. Because IL-3 as well as IL-11 are known as efficient growth factors for human megakaryocytes in vitro, our data provide insights into the regulatory mechanisms involved in megakaryopoiesis and the development of myelofibrosis.

Bone Marrow Cells↗

PubMed1994

Cytokine release by human bone marrow cells: analysis at the single cell level.

Regulation of haemopoiesis is closely mediated by a number of growth factors in the marrow microenvironment. The identification of the cell type secreting these regulatory polypeptides is difficult due to the heterogeneity of bone marrow cells. To analyse the release of haemopoietic growth factors by normal human bone marrow cells at the single cell level, we employed the reverse haemolytic plaque assay (RHPA). Freshly isolated human marrow cells were examined for the release of interleukin-1 alpha (IL-1 alpha), IL-3, IL-6 and granulocyte-monocyte colony stimulating factor (GM-CSF). In order to identify various cytokine-secreting cell types, the RHPA was combined with immunocytochemical or enzymatic staining. The total of secreting marrow cells as well as the amount of several secretory haemopoietic subpopulations could be determined with this technique under various conditions. Following incubation with pure serum-free medium without addition of any mediator, only few cells secreting either IL-1 alpha, IL-3, IL-6 or GM-CSF could be observed. After 2 h incubation with recombinant human-IL-1 alpha (rhIL-1 alpha) (10.0 ng/ml) or rhGM-CSF (10.0 pg/ml) the number of cytokine-secreting cells significantly increased for all secretory products tested. Using cytochemical staining reactions, we were able to identify 55% of all cells secreting a specific cytokine. Glycophorin C-positive erythropoietic cells turned out to be the largest fraction (up to 89%) of cytokine-releasing haemopoietic cells, followed by neutrophil granulocytes (between 6 and 48%), and monocytes/macrophages (between 4 and 23%). Only few CD 61-positive cytokine-secreting megakaryocytes could be detected. Dose- and time-dependent kinetics after stimulation with rhGM-CSF revealed that the bulk of secretory activity originates from haemopoietic or rather from erythropoietic cells following low level stimulation and after short stimulation time. Thus, our data are in keeping with the assumption, that especially erythropoietic cells are producing a repertoire of cytokines that is thought to exhibit regulatory functions within marrow microenvironment. In the present study the RHPA is presented as an appropriate tool for measuring cytokine release not only of cells of the haematopoietic system but also of other tissues, for example solid tumours or malignant lymphomas.

Bone Marrow↗

PubMed1994

DMSO respiration by the anaerobic rumen bacterium Wolinella succinogenes.

The anaerobic rumen bacterium Wolinella succinogenes was able to grow by respiration with dimethylsulphoxide (DMSO) as electron acceptor and formate or H2 as electron donors. The growth yield amounted to 6.7 g and 6.4 g dry cells/mol DMSO with formate or H2 as the donors, respectively. This suggested an ATP yield of about 0.7 mol ATP/mol DMSO. Cell homogenates and the membrane fraction contained DMSO reductase activity with a high Km (43 mM) for DMSO. The electron transport from H2 to DMSO in the membranes was inhibited by 2-(heptyl)-4-hydroxyquinoline N-oxide, indicating the participation of menaquinone. Formation of DMSO reductase activity occurred only during growth on DMSO, presence of other electron acceptors (fumarate, nitrate, nitrite, N2O, and sulphur) repressed the DMSO reductase activity. DMSO can therefore be used by W. succinogenes as an acceptor for phosphorylative electron transport, but other electron acceptors are used preferentially.

Anaerobiosis↗

PubMed1994

Angioimmunoblastic lymphadenopathy type of T-cell lymphoma and angioimmunoblastic lymphadenopathy: a clinicopathological and molecular biological study of 13 Chinese patients using polymerase chain reaction and paraffin-embedded tissues.

The morphological classification of angioimmunoblastic lymphadenopathy (AILD) or T-cell lymphoma of AILD-type (AILD-TCL) is still a subject of considerable difficulty and controversy. The aim of the current study was to examine the value of clinical, morphological, immunohistochemical variables in paraffin-embedded tissues in predicting the clonality of the respective lesion. Fifteen lymph node biopsies derived from 13 patients from Chengdu, China, were diagnosed as AILD or AILD-TCL and included in this study. The specimens were examined using a panel of monoclonal antibodies and a scoring system of morphological features. Clonality of the paraffin-embedded material was investigated using a novel polymerase chain reaction-technique to amplify rearranged T-cell receptor (TCR)-gamma sequences. Additional experiments were carried out to investigate the presence of clonal rearrangements of the immunoglobulin heavy chain (IgH) locus. We found clonal rearrangements of the TCR-gamma locus in 9 out of 15 lymph node biopsies. In 3 patients, the predominant cell clones carried clonal IgH and TCR-gamma rearrangements whereas 1 patient with polyclonal TCR-gamma pattern displayed IgH-monoclonality. The statistical evaluation of morphological and immunohistochemical data indicated that no single variable was able significantly to predict the clonality of the lesion. Furthermore, demonstrable clonality for the TCR-gamma or the IgH loci of a lesion did not correlate with a bad clinical course. Our data correlate with findings of other studies investigating AILD-TCL in Caucasian populations.

Adult↗

PubMed1994

[Determination of clonality in lymphoproliferative diseases using polymerase chain reaction].

The diagnosis of lymph node lesions frequently requires the employment of additional studies. Lineage and clonality can be regarded as the most crucial diagnostic parameters. In some cases the diagnosis remains ambiguous even after immunohistochemistry and molecular biologic techniques have to be applied. These molecular biologic techniques are based on specific rearrangements of the immune genes that occur during T- and B-cell development. Whereas Southern blotting has become a standardized and reliable means for the detection of lineage and clonality, the limited availability of fresh frozen material frequently restricts its clinical applicability. The polymerase chain reaction has facilitated the investigation of immune gene rearrangements in formalin-fixed and paraffin-embedded tissue samples. This communication reviews the recent developments in this field.

Chromosome Aberrations↗

PubMed1994

Incidence of Epstein-Barr virus bcl-2 expression and chromosomal translocation t(14;18) in large cell lymphoma associated with paragranuloma (lymphocyte-predominant Hodgkin's disease).

Seven cases of large cell lymphoma (LCL) developing simultaneously or secondarily to lymphocyte-predominant Hodgkin's disease (nodular paragranuloma [NP]) were investigated for the presence of Epstein-Barr virus genomic material and the chromosomal translocation t(14;18) involving the major breakpoint region of the bcl-2 gene using the polymerase chain reaction on paraffin-embedded material. The translocation t(14;18) and Epstein-Barr virus could not be demonstrated in any case. The expression of the bcl-2 oncogene product was investigated using immunohistochemistry. Only in one case were the lymphocytic and histiocytic cells positively stained with the bcl-2 antibody, whereas the associated LCL demonstrated a completely negative immunoreaction. In another case the LCL had a positive immunoreaction with this antibody and the corresponding lymphocytic and histiocytic cells were completely negative. We conclude that Epstein-Barr virus infection is rare or absent in NP and in the LCLs associated with NP. The chromosomal translocation t(14;18) does not seem to be a factor in the transformation of NP into LCL.

Adult↗

PubMed1994

Impact of overall treatment time of fractionated irradiation on local control of human FaDu squamous cell carcinoma in nude mice.

A series of experiments were performed to determine the impact of overall treatment time on local control of human FaDu squamous cell carcinoma irradiated with 30 fractions under ambient conditions in nude mice. The TCD50 increased with treatment time between 15 days and 10 weeks from 43 Gy to 102 Gy. The data can be well described by a single linear function. The dose recovered per day is 1.0 Gy. However, the data can also be adequately fitted by two components with an initial delay of about 30 days followed by a steep increase at a rate of 1.5 Gy per day. Assuming that the increase of TCD50 is solely caused by repopulation of clonogenic tumor cells, and that the cellular radiation sensitivity in vitro reflects the radiation sensitivity of FaDu cells in vivo, the doubling time of clonogenic tumor cells during treatment is estimated to be approximately 1.8 days for the one-component model and, after an initial delay, approximately 1.2 days for the two-component model. Both values are shorter than the doubling time of clonogenic cells in untreated FaDu tumors and similar to the potential doubling time determined by flow cytometry after BrdUrd labelling. It is concluded that the dose necessary to control FaDu squamous cell carcinoma increases considerably with increasing time of a fractionated radiation treatment. It appears most likely that this increase is caused by repopulation of clonogenic tumor cells; however, other mechanisms such as an increasing fraction of hypoxic tumor cells can not be ruled out at present.

Animals↗