Biomedical subjects
Publications and source records attributed to W Lange.
A case of bilateral microphthalmos with orbital cysts is presented with clinical, echographic, CT and genetical findings. An incomplete trisomy 22 is shown as the likewise cause of the malformation. No further symptoms known to be part of the cat-eye-syndrome could be detected.
Human megakaryocytes and platelets contain counterparts of several plasma proteins. The origin of most of these alpha-granule proteins is unclear. Fibrinogen represents one of those molecules, being essential in hemostasis, thrombosis, and platelet aggregation. To study whether fibrinogen is endocytosed by megakaryocytes and packaged into alpha-granules or newly synthesized by these cells, we established a highly sensitive nested primer polymerase chain reaction for the detection of human fibrinogen gamma-chain mRNA. In enriched megakaryocyte fractions, as well as fluorescence-activated cell sorter-purified megakaryocytes from bone marrow samples of healthy volunteers, no fibrinogen gamma-chain mRNA could be detected, despite the presence of the corresponding fibrinogen gamma-chain DNA. We conclude that fibrinogen gamma-chain mRNA, as detectable by our amplification system, is missing in megakaryocytes. This finding suggests that fibrinogen might be acquired from plasma by endocytosis and sequestered in alpha-granules before reentering the circulation after platelet activation.
The availability of sufficient quantities of recombinant human cytokines and promising preclinical data have led to their introduction into clinical trials. Cytokines have potential as new therapeutic agents in a variety of hematological disorders as well as in solid tumors. Only a few of the still increasing number of these glycoprotein hormones have been studied in humans so far, either as single agents or in combination with chemotherapy and other cytokines. Their clinical effects, beneficial role in supportive care, and use in the treatment of certain cancer patients are reviewed.
A modified two-step polymerase chain reaction (PCR) was used for the amplification of BCR/ABL mRNA in 16 patients with Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) following allogeneic bone marrow transplantation (BMT). At different intervals after BMT, patient cells were assessed for the presence of BCR/ABL mRNA by two subsequent rounds of PCR amplification; this procedure increased the sensitivity for the detection of one Ph+ cell in 10(4-5) to one cell in 10(5-6). Eight of 16 patients were negative by two-step PCR 1-39 months after BMT, suggesting an elimination of Ph-positive cells or a decrease below the threshold of detection. Although five patients showed negative results by the one-step PCR only, they were tested positive when nested primers were used, indicating a substantial decrease in the amount of BCR/ABL target mRNA compared with earlier pre- or post-transplant analyses. One patient who was still PCR positive 27 months after BMT became negative 12 months later. Persistence of BCR/ABL mRNA-expressing cells correlated with subsequent clinical relapse only when the transplantation was performed during blast crisis. All patients who underwent transplantation in chronic phase, including those with BCR rearrangement by PCR, are in clinical and hematological remission between 24 and 95 months after BMT. We conclude that aggressive chemotherapy combined with total body irradiation is unable to completely eradicate the malignant clone in all CML patients, and it might be speculated that other mechanisms (e.g., graft versus host reaction [GVHD] or graft versus leukemia effect [GVL]) may effectively eliminate residual leukemic cells.
This case report describes the clinical use of recombinant human interleukin-3 as adjunct to immunosuppressive therapy with antilymphocyte globulin, cyclosporin A, and methylprednisolone for refractory severe aplastic anemia. Hematopoietic response to treatment was moderate and peripheral blood counts (neutrophils, eosinophils, monocytes, reticulocytes) increased only slightly. Unexpectedly, during the time of interleukin-3 administration a substantial bone marrow infiltration by macrophages became detectable, consistent with the diagnosis of medullary histiocytosis, that may have prevented recovery of normal hematopoiesis in this patient. This observation may indicate the need for careful use of interleukin-3 in patients with drug-induced immunodeficiency.
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We have amplified herpes simplex virus type 1 (HSV-1) DNA sequences from individual latently infected mouse trigeminal ganglia by polymerase chain reaction (PCR) assays. This report presents two useful modifications in the PCR technique. The first involves the use of two sets of closely spaced, oppositely oriented oligonucleotide primers and two rounds of 20-40 PCR cycles, first with the more widely spaced outer primers and then with the internal nested primers. This method enhanced the sensitivity of PCR detection as shown by assays of HSV-1 sequences in human brain. The second modification was designed to detect selectively HSV-1 sense or anti-sense RNA transcripts when both are present by adding a single primer during an initial reverse-transcriptase-mediated cDNA synthesis reaction. After destruction of the RNA template, standard PCR is initiated by the addition of the second primer and thermus aquaticus DNA polymerase (Taq). We show here applications of both of these modifications to amplify HSV-1 sequences from nervous system tissue.
Studies were conducted into hormonal additives to medium and culturing time and their effects on in vitro maturation of bovine oocytes. The best maturation results were ensured in vitro by substitution of TCM-199 with FSH, HCG, and 17 beta-oestradiol: The stage of fertilisation capability (telophase I or metaphase II) was reached by 86% of all oocytes (115 of 134). The rate of maturation was worse with significance in FSH-free medium (62% or 101 of 163), and the amount of degenerated oocytes was twice as high (18%). Maturation in hormone-substituted medium for 28 to 30 hours is recommended under the condition that immature oocytes were cultured from juvenile antral follicles (2--5 mm in diameter).
Immature oocytes from antral follicles of cattle were tested for the effect of follicular factors on maturation. In vitro maturation was accomplished by use of follicular fluid from small (2--5 mm) and large (above 15 mm) follicles and by addition to the medium of a granulose factor (GF) which had been isolated from the surface of granulosa cells. The parent material, with 84% (72/86) of oocytes at the germinal vesicle stage (GV-S) at the beginning of culturing, could be rated immature. 46% of all oocytes (41/89) had reached telophase I or metaphase II (full maturation) after 24 hours of maturation in hormone-free control medium (TCM 199 + 10% of foetal calf serum). 36% of oocytes (53/84), on the other hand, stayed between GV breakdown (GVBD) and anaphase I (incipient maturation). Full maturation was reached by as little as 14%. GF and follicular fluid from small antral follicles were found to inhibit GVBD in the oocytes. 59% (36/61) or 48% (61/127) of oocytes were blocked at GV stage. Positive determination of maturation inhibiting action of the above follicular components may provide a chance for their target-oriented use in control of the maturation process. The pool of immature oocytes of the ovaries, under such circumstances, might be more systematically utilised for in vitro manipulations.
The presence, chromatographic properties and localization of neuropeptide Y was demonstrated in postmortem human brain areas of neurologically and neuropsychiatrically normative controls using immunocytochemistry and high performance liquid chromatography combined with radioimmunoassay. NPY-immunoreactivity was found in many regions of the prosencephalon. Numerous perikarya and fibers were present in the neocortex, basal ganglia and limbic-hypothalamic areas. A moderate number of neurons and fibers was observed in the basal forebrain, including the septal complex. A comparative immunohistochemical investigation in perfusion-fixed brains of the old-world ape Saguinus oedipus revealed an almost identical distribution of NPY-immunoreactivity with only minor differences. Colocalization experiments on 1-2 microns thin consecutive paraffin sections revealed a large number of NPY neurons throughout the human neostriatum and amygdaloid complex that were also positive for somatostatin. Our findings indicate that detection of neuropeptides in fresh or fixed post-mortem human tissue by different immunochemical methods may actually reflect the in vivo conditions. In addition, the wide distribution of NPY throughout the human brain and its colocalization with other neurotransmitters suggests a physiological role as neuroactive substance, i.e. neuromodulator in the primate central nervous system.
An immunohistochemical study in which antisera against several neuropeptides were used demonstrated the presence of neuropeptide Y(NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivity in nerve fibers in the human prostate gland and seminal vesicle, whereas no immunostaining for substance P and calcitonin gene-related peptide was observed. The peptidergic innervation was found to be generally moderate to low. NPY- and VIP-immunoreactive fibers were localized in the subepithelial connective tissue as well as the smooth muscle layers in both organs, although the peptidergic fiber networks were more prominent in the seminal vesicle. Most NPY-immunoreactive fibers were observed in the musculature of the seminal vesicle. In addition, NPY- and VIP-immunoreactive fibers were demonstrated in the walls of blood vessels. The results of our study suggest that the innervation of the prostate gland and seminal vesicle by various neuroactive peptides may be involved in the autonomic regulation of these organs in adult man, as well as sympathetic and parasympathetic nerve fibers.
The presence of atrial natriuretic peptic/cardiodilatin-immunoreactive material was demonstrated in the lacrimal gland of the domestic pig by high performance liquid chromatography and radioimmunoassay. The immunohistochemical localization revealed a distinct population of cuboid or spindle-shaped ANP/CDD-IR cells in the epithelium of the terminal portion of the secretory tubules. In addition, a moderate number of positive cells was localized intraepithelially in the intralobular ducts as well as the connective tissue between these ducts. Our findings provide a morphological indication that ANP/CDD may play a physiological role in the regulation of sodium transport and secretion in the lacrimal gland.
Adamantylamide dipeptide (AdDP) is a novel synthetic compound combining the antiviral properties of amantadine and the essential adjuvant activity of immunomodulator muramyl dipeptide. Mice were immunized with influenza A/Taiwan/1/86 (H1N1), A/Sichuan/2/87 (H3N2) and influenza B/Beijing/1/87 subunit vaccines containing AdDP or aluminium hydroxide (Al(OH)3). Induction of homologous haemagglutination-inhibition (HI) antibodies and correlation to protection against lethal aerosol influenza A/PR/8/34 (H1N1) infection were investigated. Subunit vaccine containing A/Sichuan (H3N2) and Al(OH)3 stimulated high HI antibody titres but failed to provide protection against heterologous influenza A (H1N1) challenge infection following either the primary or the secondary immunizations. In contrast, similar treatment with A/Sichuan subunit vaccine containing AdDP conferred significant protection against heterologous challenge despite low levels of circulating antibody. Primary immunization with even influenza B/Beijing subunit vaccine containing AdDP, but not Al(OH)3, provided partial protection against influenza A challenge. These results suggest that appropriate immunomodulators like AdDP can convert restricted homotypic immunity induced by inactivated influenza subunit vaccines to advantageous cross-reacting type of heterologous response.
In the search for compounds capable of inducing endogenous production of colony-stimulating factor (CSF) and possessing activity against human immunodeficiency virus (HIV), an immunomodulator, muramyl dipeptide (MDP), was investigated. MDP can enhance monocyte-macrophage CSF in serum and promote nonspecific resistance against a variety of microbial pathogens. MDP exhibited an inhibitory activity against HIV infection of CD4+ H9 lymphocytes and U937 monocytoid cells. An inhibitor of viral reverse transcriptase, 2', 3'-dideoxyadenosine, produced potent inhibition in cultures which were similarly infected with HIV. MDP could partially reduce antigen production in persistently HIV-infected KE37/1 lymphocyte cultures.
Common bacterial infections are increasingly being diagnosed in HIV-infected individuals. Cells of the monocyte-macrophage lineage kill invading bacterial pathogens and subsequently release immunoadjuvant components from the degraded cell walls. Since monocytes can be infected with HIV, effects of bacterial immunomodulators on infected promonocytic U937 cells were investigated. Synthetic muramyl peptide, mycobacterial trehalose dimycolate, and detoxified endotoxin exhibited an initial reduction followed by a rapid increase in HIV p24 antigen production. The upregulation of virus expression was correlated with enhanced interleukin-1 beta levels and a decrease in TNF-alpha production.
There are notable interspecific differences in the lectin binding sites in the retina of mammals. In the porcine retina a distinct labeling of all the neurons of the stratum ganglionare is obtained by use of succinylated wheat-germ-agglutinin (WGA-S). In the baboon the stratum ganglionare does not label, though the entire inner plexiform layer can be demonstrated with WGA-S. In the cat WGA-S binds exclusively to the outer segments of the rods and cones. These specific differences will be compared with findings in the human retina [10] and discussed in connection with biochemical results obtained during our studies of the porcine retina.
The effect of the cumulus oophorus on fertilisation was investigated by 478 in vitro maturated bovine oocytes. Culturing revealed that the amount of defects on the pellucid zone and in ooplasm of cumulus-free oocytes was higher than that in oocytes cultured in the cumulus-oocyte complex (38 percent or 77/203 versus eleven percent or 30/275). Insemination of the cumulus-oocyte complex (COC) seemed to increase the fertilisation capability of bovine oocytes (63 percent or 70/111), though its penetration rate was only 45 percent (111/245) and was thus below that of cumulus-free oocytes (55 percent or 69/126). Use of COC is recommended as basic material for in vitro maturation and fertilisation.
The Philadelphia chromosome of chronic myelogenous leukemia (CML) patients is caused by a translocation of the c-abl gene from chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22. A new bcr-abl mRNA is expressed in these cases. We have developed a modified polymerase chain reaction (PCR) for the detection of this mRNA. The method is extremely sensitive, reliable, and relatively fast. The analysis of peripheral blood or bone marrow cells from CML patients treated with chemotherapy shows that the two possible mRNAs are expressed in various combinations. Our results show that even after myeloablative therapy for bone marrow transplantation bcr-abl mRNAs are still expressed. Further studies, however, are necessary to determine the clinical relevance of a small number of persisting cells expressing the bcr-abl mRNA.