Postmortem Diagnosis of Acute Megakaryocytic Leukemia Usefulness of lmmunohistochemistry and Tissue Hemogram

An autopsy case of acute megakaryocytic leukemia (AMKL) is presented. The bone marrow was hypercellular with proliferation of three lineages, especially megakaryocytes. Immunohistochemical examination revealed many platelet glycoprotein IIb/IIIa (GP IIb/IIIa)- positive blast cells in bone marrow. The proportion of the blasts was 26.4% by tissue hemogram. GP IIb/IIIa-positive blasts and megakaryoblasts were deposited massively in lymph nodes. lmmunohistochemistry against GP IIb/IIIa and tissue hemograms by paraffin section are needed to diagnose AMKL by postmortem examination, since the identification of ultra-structural platelet peroxidase in autopsy materials is difficult.     

Dissociation of interleukin-2 production from the cell activation in response to the mitogenic lectin in peripheral CD4+ T cells of LEC mutant rats.

We have recently shown that an exogenous gradient of interleukin-8 (IL-8) induces the transendothelial migration of neutrophils. Treatment of endothelium with the cytokines IL-1 or tumour necrosis factor (TNF) also causes neutrophil transmigration, and recent evidence suggests that this may be due to endogenous IL-8 produced by the endothelium. We have used specific chemotactic desensitization of neutrophils to investigate the role of IL-8 in transmigration through cytokine-activated endothelium. Preincubation of neutrophils with IL-8 reduced their chemotactic transmigration response to an IL-8 gradient by 81%, demonstrating desensitization. Transmigration in response to cytokine-activated endothelium was inhibited by 104% after IL-8 preincubation, thus tending to support the role of IL-8. However, preincubation with another neutrophil chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (FMLP), which did not affect the IL-8, response, also inhibited transmigration, by 74%. This suggests that FMLP preincubation acts to inhibit a non-IL-8-dependent mechanism of transmigration through cytokine-activated endothelium. Chemotactic factor pretreatment of neutrophils did not reduce their adhesion to activated endothelium, but specifically blocked the transmigration step. We have therefore shown that chemotactic transmigration can be subjected to factor-specific desensitization, and have used this to provide evidence supporting a role for IL-8 in transmigration through cytokine-activated endothelium, as well as suggesting a further IL-8-independent mechanism. These data also provide a mechanism for the observed defect in accumulation of neutrophils at inflammatory sites when chemotactic factors are infused intravenously.     

Frequent co-localization of Cox-2 and laminin-5 gamma2 chain at the invasive front of early-stage lung adenocarcinomas

Laminin-5 is an extracellular matrix protein that plays a key role in cell migration and tumor invasion. Cox-2 is an induced isoform of cyclooxygenases that plays an important role in carcinogenesis, suppression of apoptosis, angiogenesis, and metastasis of colon cancer. We report frequent co-expression of cox-2 and laminin-5 at the invasive front of early-stage lung adenocarcinomas. We investigated the expression of cox-2 and laminin-5 immunohistochemically in 102 cases of small-sized lung adenocarcinoma (maximum dimension, 2 cm or less). Cox-2 and laminin-5 were expressed in 97 (95.1%) and 82 (80.4%) cases, respectively. Both were preferentially localized in cancer cells at the cancer-stroma interface, although cox-2 tended to show a diffuse staining pattern in some cases. A comparison of their staining patterns revealed a striking similarity in their distribution in 24 cases, and a partial overlap between their localization in another 20 cases. Moreover, an overall correlation was found between the expression levels of cox-2 and laminin-5 (P = 0.018). To gain insight into the mechanisms that regulate the expression of these proteins, we additionally studied their expression in 58 cases of stage I lung adenocarcinoma, in which p53 status was determined by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism analysis, and direct sequencing. The results showed that tumors with mutant p53 tended to express more cox-2 than those with wild-type p53 (P = 0.080). Also, tumors that overexpressed p53 had higher levels of cox-2 and laminin-5 than those without p53 overexpression (P = 0.032 and 0.047, respectively). Further immunohistochemical analysis showed that tumors that overexpressed both epidermal growth factor receptor (EGFR) and erbB-2 had higher levels of cox-2 and laminin-5 than those without concomitant overexpression of these proteins (P = 0.014 and P = 0.018, respectively). To see whether EGFR signaling is involved in cox-2 and laminin-5 expression, we further conducted in vitro analyses using six lung adenocarcinoma cell lines (A549, HLC-1, ABC-1, LC-2/ad, VMRC-LCD, and L27). Western blot analyses showed that cox-2 mRNA levels, and to a lesser extent laminin-5 gamma2 mRNA levels, correlated with the expression levels of erbB-2 and the phosphorylated form of MAPK/ERK-1/2 protein. The addition of transforming growth factor-alpha increased both cox-2 and laminin-5 gamma2 mRNA levels in A549, ABC-1, and L27 with different kinetics; the induction of cox-2 occurred earlier than that of laminin-5 gamma2. Finally, the migration of ABC-1 cells was inhibited by MAP kinase kinase inhibitor PD98059 and a selective cox-2 inhibitor NS-398. In contrast, the migration of A549 cells was inhibited by PD98059, but much less effectively by NS-398. These results suggest that co-stimulatory mechanisms may exist that increase the expression of cox-2 and laminin-5 at the invasive front of lung adenocarcinomas and that EGFR signaling could be one of the mechanisms. Further investigations are warranted concerning the role of cox-2 and laminin-5 in cancer cell invasion and the significance of p53 and EGFR signaling in the regulation of cox-2 and laminin-5 expression.     

Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization

Retinal vasculogenesis and ischemic retinopathies provide good model systems for study of vascular development and neovascularization (NV), respectively. Vascular endothelial cell growth factor (VEGF) has been implicated in the pathogenesis of retinal vasculogenesis and in the development of retinal NV in ischemic retinopathies. However, insulin-like growth factor-I and possibly other growth factors also participate in the development of retinal NV and intraocular injections of VEGF antagonists only partially inhibit retinal NV. One possible conclusion from these studies is that it is necessary to block other growth factors in addition to VEGF to achieve complete inhibition of retinal NV. We recently demonstrated that a partially selective kinase inhibitor, PKC412, that blocks phosphorylation by VEGF and platelet-derived growth factor (PDGF) receptors and several isoforms of protein kinase C (PKC), completely inhibits retinal NV. In this study, we have used three additional selective kinase inhibitors with different selectivity profiles to explore the signaling pathways involved in retinal NV. PTK787, a drug that blocks phosphorylation by VEGF and PDGF receptors, but not PKC, completely inhibited retinal NV in murine oxygen-induced ischemic retinopathy and partially inhibited retinal vascularization during development. CGP 57148 and CGP 53716, two drugs that block phosphorylation by PDGF receptors, but not VEGF receptors, had no significant effect on retinal NV. These data and our previously published study suggest that regardless of contributions by other growth factors, VEGF signaling plays a critical role in the pathogenesis of retinal NV. Inhibition of VEGF receptor kinase activity completely blocks retinal NV and is an excellent target for treatment of proliferative diabetic retinopathy and other ischemic retinopathies.     

Characterization of the UL4 gene product of herpes simplex virus type 2

Summary.  We have identified the herpes simplex virus type 2 (HSV-2) UL4 gene product using a rabbit polyclonal antiserum raised against a recombinant 6xHis-UL4 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 27-kDa protein in HSV-2 186-infected cell lysates. The protein was not detectable in the presence of the viral DNA synthesis inhibitor, suggesting that the UL4 gene was expressed as a γ₂ gene. Indirect immunofluorescence studies localized the UL4 protein within the nucleus as discrete punctate forms at late times postinfection. However, when expressed in the absence of other viral proteins, the UL4 protein was limited to the cytoplasm, indicating that an interaction with one or more other virus-induced proteins was responsible for the nuclear localization during infection. Subnuclear fractionation studies showed that the protein was released from the nuclear structure of infected cells by high salt treatment. Moreover, the UL4 protein was detected in purified virions and light particles. Received December 24, 1997 Accepted February 4, 1998     

Genitourinary phenotype in XX patients with distal 9p monosomy

Although testicular development has been shown to be variably impaired in XY patients with distal 9p monosomy, ovarian and other genitourinary phenotype has poorly been studied in XX patients monosomic for the distal 9p region. Thus, we studied a 13-month-old infant with 46,XX,der(9)t(9;10)(p23;p13) (case 1) and an 11-year-old girl with 46,XX,der(9)t(9;16)(p23;q22) (case 2). Case 1 had primary hypogonadism (basal serum follicle stimulating hormone [FSH], 40.0 mIU/mL; leteinizing hormone [LH], 1.2 mIU/mL; estradiol [E2], <10 pg/mL), whereas case 2 had age-appropriate pubertal development (breast, Tanner stage 4; pubic hair, Tanner stage 3; menarche 11.7 years of age) and hormone values (FSH, 7.3 mIU/mL; LH, 6.7 mIU/mL; E2, 47 pg/mL). In addition, case 1 had hypoplastic labia majora, short distance between the vaginal orifice and the anus, and five renal cysts, and case 2 had anal atresia, short distance between the vaginal orifice and the anus, bilateral hydronephrosis of grade 3 with probable ureteropelvic junction stenosis, and renal dysfunction (serum creatinine, 1.52 mg/dL; urea nitrogen, 34.5mg/dL). Fluorescence in situ hybridization analysis for five regions and microsatellite analysis for 10 loci on 9p confirmed hemizygosity for the distal 9p region with the breakpoints between IFNA and D9S285 in case 1 and between D9S168 and D9S286 in case 2. The results, in conjunction with the previous data in XX patients with molecularly defined distal 9p monosomy, are consistent with the presence of a gene(s) involved in the development of indifferent gonad or subsequent ovarian differentiation in a approximately 11 Mb region distal to D9S168. In addition, it is possible that a gene(s) for anoperineal and renal development also maps distal to D9S168 and that for external genital development maps distal to D9S285 at the position approximately 16 Mb from the 9p telomere.     

Propagation of action potentials from the soma to individual dendrite of cultured rat amacrine cells is regulated by local GABA input

Retinal amacrine cells are interneurons that make lateral and vertical connections in the inner plexiform layer of the retina. Amacrine cells do not possess a long axon, and this morphological feature is the origin of their naming. Their dendrites function as both presynaptic and postsynaptic sites. Half of all amacrine cells are GABAergic inhibitory neurons that mediate lateral inhibition, and their light-evoked response consists of graded voltage changes and regenerative action potentials. There is evidence that the amount of neurotransmitter release from presynaptic sites is increased by spike propagation into the dendrite. Thus understanding of how action potentials propagate in dendrites is important to elucidating the extent and strength of lateral inhibition. In the present study, we used the dual whole cell patch-clamp technique on the soma and the dendrite of cultured rat amacrine cells and directly demonstrated that the action potentials propagate into the dendrites. The action potential in the dendrite was TTX sensitive and was affected by the local membrane potential of the dendrite. Propagation of the action potential was suppressed by local application of GABA to the dendrite. Dual dendrite whole cell patch-clamp recordings showed that GABA suppresses the propagation of action potentials in one dendrite of an amacrine cell, while the action potentials propagate in the other dendrites. It is likely that the action potentials in the dendrites are susceptible to various external factors resulting in the nonuniform propagation of the action potential from the soma of an amacrine cell.     

Expression of epithelial adhesion proteins and integrins in chronic inflammation.

Epithelial cell behavior in chronic inflammation is poorly characterized. During inflammation of tooth-supporting structures (periodontal disease), increased proliferation of epithelial cells into the inflamed connective tissue stroma is commonly seen. In some areas ulceration and degeneration take place. We studied alterations in the expression of adhesion molecules and integrins during chronic periodontal inflammation. In inflamed tissue, laminin-1 and type IV collagen were still present in the basement membrane and surrounding blood vessels, but they were also found extravascularly in inflamed connective tissue stroma. Type VII collagen and laminin-5 (also known as kalinin, epiligrin, or nicein) were poorly preserved in the basement membrane zone, but both were found in unusual streak-like distributions in the subepithelial connective tissue stroma in inflamed tissue. Both fibronectin and tenascin were substantially decreased in chronically inflamed connective tissue, showing only punctate staining at the basement membrane zone. Integrins of the beta 1 family showed two distinct staining patterns in epithelial cells during chronic inflammation; focal losses of beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1) were found in most areas, while in other areas the entire pocket epithelium was found to be strongly positive for beta 1 integrins. No members of the alpha v integrin family were found in any epithelia studied. Expression of the alpha 6 beta 4 integrin was high in basal cells of healthy tissue, but weak in epithelium associated with chronic inflammation. Chronic inflammation therefore involves alterations in both adhesion proteins and integrins expressed by epithelial cells. Basement membrane components found at abnormal sites in stroma in chronic inflammation might serve as new adhesive ligands for various cell types in inflamed stroma.     

Intracellular pH measurement in frog muscle by means of 31P-nuclear magnetic resonance.

The 31P-NMR technique was used for the monitoring of intracellular pH and studying its heterogeneity in the femoral biceps muscle of Rana catesbiana under anaerobic conditions. The value of intracellular pH of fresh muscle calculated from the chemical shift of intracellular inorganic phosphate (P1) was 7.3 on average and the line width of P1 was about 0.2 ppm. As the line width determined by the relaxation mechanism was 0.099 ppm, the P1 signal in fresh muscle was concluded to consist of overlapped narrow components, which indicated the heterogeneity of muscular pH (about 0.2 pH unit). Living muscle showed gradual acidification due to glycolysis and the decrease in heterogeneity. When glycolysis was suppressed by iodoacetic acid, slight alkalization due to the breakdown of creatine phosphate was observed. When the Lohmann reaction was suppressed by 2, 4-dinitro-1-fluorobenzene, rapid acidification accompanied by the appearance of a new acidic component was observed with the onset of ATP decrease. This new component was not detected in the muscle pretreated with glycerol to disrupt the transverse tubules. Therefore, it is likely that this new acidic component originates in the intracellular compartment, and not in the cellular difference.     

IgE antibody responses induced by transplantation of the nematode Nippostrongylus brasiliensis in rats: a possible role of nematode excretory-secretory product in IgE production.

In order to examine the effective site of sensitization for IgE responses, we transplanted 2000 adult-stage worms of the nematode Nippostrongylus brasiliensis into the duodenum or the peritoneal cavity of naive rats. Total serum IgE began to increase 1 week after the nematode inoculations and reached a peak at week 2. Living worms inoculated into the duodenum induced the highest serum IgE, this being 800 times the level in control animals. Intraperitoneal inoculations of living and dead worms resulted in increases of the serum IgE levels to 120 and 13 times the control level, respectively. The intraduodenal inoculation of living adult worms also induced a significant increase in specific IgE against the excretory-secretory (ES) product of adult N. brasiliensis 1 week later than the rise in total IgE, whereas intraperitoneal inoculations did not induce such an increase. These results suggest that sensitization through the intestinal mucosa with adult N. brasiliensis might be important for the effective induction of both specific and non-specific IgE responses. Since these findings also indicated that factors secreted by living worms play an important role in the induction of total IgE response, the ES product was injected to naive rats for 6 consecutive days (total 2.7-4.4 mg). Intraperitoneal injection of the ES product alone induced a 14.7-fold increase in total IgE without any specific IgE response. This indicates that some constituents of the ES product have the potential to trigger a non-specific IgE response.