Characteristics, outcomes, prognostic factors and treatment of patients with T-cell prolymphocytic leukemia (T-PLL).

BACKGROUND: T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive disease. In this study, we report our experience from 119 patients with T-PLL. PATIENTS AND METHODS: We reviewed the clinico-pathologic records of 119 consecutive patients with T-PLL, who presented to our institution between 1990 and 2016. RESULTS: One hundred and nineteen patients with T-PLL were analysed. Complex karyotype and aberrations in chromosome 14 were seen in 65% and 52% patients, respectively. Seventy-five patients (63%) were previously untreated and 43 (37%) were initially treated outside our institution. Sixty-three previously untreated patients (84%) received frontline therapies. Overall, 95 patients (80%) have died. Median overall survival (OS) from diagnosis was 19 months [95% confidence interval (CI) 16-26 months]. Using recursive partitioning (RP), we found that patients with hemoglobin < 9.3 g/dl, lactate dehydrogenase (LDH) ≥ 1668 IU/l, white blood cell ≥ 208 K/l and ß2M ≥ 8 mg/l had significantly inferior OS and patients with hemoglobin < 9.3 g/dl had inferior progression-free survival (PFS). In multivariate analysis, we identified that presence of pleural effusion [hazard ratio (HR) 2.08 (95% CI 1.11-3.9); P = 0.02], high LDH (≥ 1668 IU/l) [HR 2.5 (95% CI 1.20-4.24); P < 0.001)], and low hemoglobin (< 9.3 g/dl) [HR 0.33 (95% CI 0.14-0.75); P = 0.008] were associated with shorter OS. Fifty-five previously untreated patients received treatment with an alemtuzumab-based regimen (42 monotherapy and 13 combination with pentostatin). Overall response rate, complete remission rate (CR) for single-agent alemtuzumab and alemtuzumab combined with pentostatin were 83%, 66% and 82%, 73% respectively. In patients who achieved initial CR, stem cell transplantation was not associated with longer PFS and OS. CONCLUSION: Outcomes in T-PLL remain poor. Multicenter collaborative effort is required to conduct prospective studies.

Effect of AA861, a 5-lipoxygenase inhibitor, on amylase secretion from rat pancreatic acini.

We have examined the effects of 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), a selective inhibitor of 5-lipoxygenase, on the action of cholecystokinin (CCK) and other secretagogues in the stimulation of amylase secretion from dispersed rat pancreatic acini. AA861 inhibited amylase secretion caused by CCK, carbamylcholine (carbachol), bombesin or calcium ionophore A23187 but failed to affect amylase secretion by vasoactive intestinal peptide or 12-O-tetradecanoyl-phorbol 13-acetate. Inhibition by AA861 of CCK or carbachol-induced amylase secretion was confined to the relatively lower concentrations of these secretagogues. AA861 did not inhibit receptor binding of CCK or alter the cellular calcium mobilization induced by CCK. In kinetic studies, AA861 was effective only on amylase secretion from pancreatic acini incubated with CCK for more than 5 min. Indomethacin, a known inhibitor of cyclooxygenase, did not affect the amylase secretion caused by all secretagogues used. These results indicate that the 5-lipoxygenase pathway of arachidonate metabolism may be involved in the actions of calcium-dependent secretagogues of amylase secretion in rat dispersed pancreatic acini, especially for sustaining stimulation of amylase secretion by CCK.

Clonality analysis by methylation-specific PCR for the human androgen-receptor gene (HUMARA-MSP).

The human androgen-receptor gene (HUMARA) has been used for analysis of X chromosome inactivation (XCI) pattern because of a polymorphic short tandem repeat (STR) near the 5'-promoter region correlated with XCI. We introduce a novel method to analyze XCI pattern, named HUMARA methylation-specific PCR (HUMARA-MSP) assay, which analyzes methylation status of the HUMARA gene by bisulfite modification instead of a methylation-sensitive restriction enzyme. Although the original MSP method shows whether there is a methylated band or not, our HUMARA-MSP method identifies the patterns of methylated and unmethylated bands. Because this method identifies either unmethylated or methylated alleles in each PCR tube and shows opposite band patterns dependent on methylation status, we can assess the XCI pattern independently twice. This method can avoid false results by incomplete enzyme digestion and incomplete bisulfite modification will not affect the results. Extremely small quantities of samples, such as hematopoietic colonies, were also available for HUMARA-MSP assay. Because DNA modified by sodium bisulfite is also available for assessment of methylation status of other genes by setting specific primers for them, we performed the simultaneous assessment of clonality and aberrant hypermethylation of p15INK4B gene in myelodysplastic syndromes. These simultaneous assessments were easily possible and provided much information despite requiring only a small volume of DNA. The HUMARA-MSP assay may facilitate the analyses for pathogenesis of hematological disorders because of its simplicity, sensitivity and wide applicability. Leukemia (2000) 14, 207-212.

Methylation status of the p15INK4B gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes.

We previously reported that the hypermethylation of the p15INK4B gene promoter was frequently observed in myelodysplastic syndromes (MDS), and that it may be associated with disease progression. An unanswered question is whether p15INK4B gene methylation is restricted to undifferentiated blastic cells, or whether differentiated cells such as granulocytes or erythrocytes of MDS origin also harbor this epigenetic alteration. In this study, we analyzed the methylation status of the p15INK4B gene in MDS by the methylation-specific PCR (MSP) method, which is more sensitive than Southern blotting. The bone marrow mononuclear cells (BM-MNCs) of 23 MDS patients were analyzed, and six of them showed p15INK4B methylation. Progenitor assay with methylcellulose medium was also performed in all patients. In two of the six patients with p15INK4B-methylated BM-MNCs, erythroid and/or non-erythroid colonies formed were subjected to molecular analysis. Colonies with and without p15INK4B methylation were detected in both patients. Furthermore, X-chromosome inactivation (XCI) pattern of each colony was simultaneously determined by MSP-based human androgen receptor gene analysis (HUMARA-MSP), and all p15INK4B-methylated colonies showed the same XCI pattern, which was dominant among the colonies, while p15INK4B-unmethylated colonies showed both patterns of XCI, in each of the two patients. We then examined the methylation status of the p15INK4B gene of granulocyte (PB-PMN) fractions from 10 patients with available peripheral blood cells. In all four patients with p15INK4B-methylated BM-MNCs, their PB-PMNs showed p15INK4B methylation. These results suggest that p15INK4B methylation in hematopoietic cells in MDS patients is restricted to the MDS clone but not necessarily to blast cells.

Purification of specific antibody against aspartate and immunocytochemical localization of aspartergic neurons in the rat brain.

The distribution of L-aspartate known as a putative excitatory neurotransmitter in the central nervous system was investigated immunocytochemically in the rat brain. Anti-aspartate antiserum was raised in rabbits using L-aspartate covalently conjugated to rabbit serum albumin with glutaraldehyde as the immunogen and was found to be cross-reactive with an L-glutamate conjugate. Monospecific anti-L-aspartate antibody was successfully purified using affinity gels coupled with several amino acids including L-aspartate and L-glutamate and with the L-glutamate conjugate. Putative aspartergic neurons were generally immunoreactive to the purified antibody, but epithelia of the choroid plexus were also stained. These results show that the antibody is a useful tool for the immunocytochemical demonstration of possible aspartergic neurons in the central nervous system, although the immunochemical expression of L-aspartate not used as a neurotransmitter must be taken into consideration.

Distribution of OL-protocadherin protein in correlation with specific neural compartments and local circuits in the postnatal mouse brain.

OL-protocadherin (OL-pc) is a cell adhesion molecule that belongs to the cadherin superfamily. A previous study showed that expression of OL-pc mRNA was specific to certain brain nuclei including those of the olfactory and limbic systems, thus suggesting its involvement in neural circuit formation. Here, we examined the distribution of OL-pc protein in the postnatal mouse brain by immunohistochemistry to confirm the possibility of such a role. The results showed that the protein could be mapped to many brain compartments including brain nuclei and higher subdivisions as previously observed for the expression pattern of the mRNA. Sharp boundaries of the distribution were often seen in areas such as the interpedunclar nucleus, cerebellar cortex, and inferior olive. In addition, the protein was detected in some fibers that could not be examined by the previous study using in situ hybridization. For example, prominent staining was noted in the stria medularis, stria terminalis, fasciculus retroflexus, optic tract, and inferior thalamic radiation, structures that seem to connect OL-pc-positive brain regions. These OL-pc-positive brain nuclei and fiber tracts coincide with some local circuits of functional systems such as the olfactory system, nigrostriatal projection, olivo-cerebellar projection, and visual system. These results support the possibility that OL-pc is involved in the formation of specific neural compartments and circuits in the developing brain.

An immunohistochemical study of aspartate, glutamate, and taurine in rat kidney.

Biochemical studies have revealed considerable amounts of free amino acids in the kidney. We examined the intrarenal distribution of three amino acids (aspartate, glutamate, and taurine) in the rat kidney with an immunoperoxidase method. In the renal cortex, all three amino acids were concentrated in the renal corpuscles and in the epithelia of the collecting tubules. Immunostaining of the collecting tubules was more intense in the principal cells than in the intercalated cells. The distal convoluted tubules were also immunostained with aspartate- and glutamate- specific antibodies but not with the taurine-specific antibody. In the renal medulla, the immunoreactivity specific for aspartate and for glutamate was similar; it was weak in the thick portion of the loop of Henle and strong in the collecting tubules. Immunoreactivity specific for taurine was restricted to regions within the epithelia of the thin portion of the loop of Henle and the collecting tubules. The significance of the accumulated amino acids as osmoregulatory agents is discussed.

Immunohistochemical localization of arginine and citrulline in rat renal tissue.

Using antibodies highly specific for L-arginine and L-citrulline, we localized these amino acids in rat kidney with immunohistochemical methods. Highest levels of arginine immunoreactivity were observed in epithelial cells of proximal tubules in the outer stripe of the outer medulla and the collecting ducts in the cortex. Staining intensity of proximal convoluted tubules in the outer stripe decreased from the inner side to the outer side. In the inner medulla, collecting ducts were labeled with moderate intensity. Staining within the cortex was apparent only with collecting ducts. Citrulline immunoreactivity was localized in the epithelial cells of collecting ducts both in the cortex and medulla. Immunoreactivity was also found in glomerular podocytes and in the epithelial cells of proximal convoluted tubules in the outer medulla. These localizations were different from those of other amino acids previously reported. The precise cellular distribution of arginine and citrulline in rat kidney was determined for the first time by an immunohistochemical method in the present study.

Septal perfusion and wall thickening in patients with left bundle branch block assessed by technetium-99m-sestamibi gated tomography.

UNLABELLED: Septal hypoperfusion is often observed in patients with complete left bundle branch block (LBBB) in myocardial perfusion imaging. Abnormal wall motion in the septal region may potentially cause artifactual perfusion abnormalities. To assess the effect of abnormal wall thickening on myocardial perfusion images, ECG-gated sestamibi SPECT was performed on 12 patients with LBBB and 10 normal subjects used as controls. METHODS: After administration of 740 MBq 99mTc-sestamibi injection at rest, ECG-gated SPECT was obtained 60 min later with division of the cardiac cycle into eight frames. RESULTS: Septal hypoperfusion was noted in 10 patients on nongated images and 11 patients on end-systolic (ES) images, whereas only two patients showed abnormalities on end-diastolic (ED) images. The septal to lateral wall count ratio in the LBBB group was lower (0.72 +/- 0.09) than in the control group (0.84 +/- 0.09) (p < 0.01) at nongated images, while it was similar at ED images (0.84 +/- 0.11 versus 0.86 +/- 0.12; ns). In addition, the count increase from ED to ES during a cardiac cycle in the septal region was smaller compared with the lateral region in the LBBB patients (25% +/- 19% in the septal region, versus 48% +/- 14% in the lateral region; p < 0.01), indicating less wall thickening in the septal region. CONCLUSION: Smaller count increase due to reduced wall thickening in the septal region may mimic hypoperfusion in patients with LBBB. This artifact can be eliminated with ECG-gated 99mTc-sestamibi SPECT, particularly with ED images.

Human pancreas scintigraphy using iodine-123-labeled HIPDM and SPECT.

The pancreatic affinity of iodine-123-labeled HIPDM (N,N,N'-trimethyl-N'-(2-hydroxy-3-methyl-5-iodobenzyl)-1,3-propane diamine) ([123I]HIPDM) was studied in 18 cases (5 normal volunteers, 7 cases with pancreas cancer, and 6 with chronic pancreatitis). In the normal cases, the pancreas was visualized in the planar images as early as 3 hr, and again at 20 hr postinjection. Single-photon emission computed tomography (SPECT) performed following 3-hr planar scintigraphy, provided excellent pancreas images without an overlap of activity in the liver or spleen. The mean pancreas-to-liver (P/L) ratio was 1.26 +/- 0.22 in normal controls. With the exception of one case of massive calcification in the pancreas, the entire pancreas could be observed in the cases with chronic pancreatitis, but the P/L ratio was 0.74 +/- 0.15, significantly lower than that of normal cases. Defective areas of the distal portion of the pancreas were clearly seen in those with cancer of the pancreas. The results of our study indicate that [123I] HIPDM may have clinical potential as a human pancreas imaging agent.

Postnatal changes of the number and lobular distribution of acid phosphatase positive and lipid granule-containing cells in the cerebellum of hyperbilirubinemic Gunn rats.

Developing homozygous (jj) and heterozygous (j+) Gunn rat cerebella were examined histologically from postnatal days 5 to 60. Sagittal sections of the cerebellar vermis of jj rats revealed that the anterior and medial lobes were significantly smaller in area than in j+ rats on and after postnatal day 10. However, the nodulus did not display significant differences in jj rats. Two classes of acid phosphatase (ACPase)-positive cells (L cells and S cells), and lipid granule-containing S cells were recognized exclusively in the jj rat cerebellum during the postnatal period studied. S cells, which are probably microglia, had an oval dark nucleus and contained many lysosomes, some of which contained lipid droplets. They appeared in all the lobules except the nodulus on day 5 and reached maximum in incidence by day 15. They were distributed all over the cerebellar layers including the white matter. Lipid granule-containing S cells appeared on postnatal day 10 and were most abundant in severely affected lobules, such as the declive and tuber, on day 30. Purkinje cells of jj rats showed vacuolation in their cytoplasm on and after postnatal day 5. After 20 days of life, the number of Purkinje cells in anterior- and medius-lobus were markedly decreased. Some severely damaged Purkinje cells became L cells with an extremely high ACPase activity. They appeared initially on postnatal day 15 and increased in number until day 30. No L cells were observed in the nodulus. These show that the severely damaged Purkinje cells and ACPase-positive and lipid granule-containing microglia cells are most abundant in the late- and intermediate-maturing regions of the vermis. Since they are either rare or absent in any earlier-maturing region, the nodulus, these data suggest toxic effects of bilirubin in the cerebellum are closely related to the ontogenic development of the individual cerebellar lobules.

Spontaneous hemopneumothorax.

BACKGROUND: Spontaneous hemopneumothorax is a rare disorder, occurring in 1% to 12% of patients with spontaneous pneumothorax. We studied our previously treated patients to determine the nature of optimal operative management. METHODS: This was a retrospective case study. From 1987 to 1994, of 428 cases of spontaneous pneumothorax that occurred in 234 patients treated at our institution, hemopneumothorax developed in 10 patients (2.3%). The clinical features of these patients were studied. RESULTS: The amount of bleeding ranged from 600 to 1,600 mL, and 3 patients exhibited symptoms of shock, such as sweating, nausea, and syncope. Six patients underwent operation within 7 days from the onset, and this involved resection of the bullae or pneumorrhaphy, or both. The source of bleeding was identified in 5 patients. Pathologic examination showed marked fibrosis with alcian blue-positive deposits of aberrant vessels. All 6 patients continue to be well postoperatively without recurrence or complications. Four patients did not undergo early thoracotomy. However, decortication was required in 3 of these patients because of a reactive fluid collection in the pleural space, which led to impaired lung expansion. CONCLUSIONS: Early surgical repair should be considered once diagnosis of a spontaneous hemopneumothorax is confirmed, because this provides better long-term results. Video-assisted thoracoscopic surgery as well as minithoracotomy should be considered as surgical options because of the improved quality of life they confer.

2'-3'-cyclic nucleotide 3'-phosphohydrolase activity in the central nervous system of a myelin deficient mutant (Shiverer).

Shiverer is a recessive autosomal mutant characterized by the deficiency of the myelin in the central nervous system. 2',3'-Cyclic nucleotide 3'-phosphohydrolase (CNPase) activity of the various parts of the central nervous system from the Shiverer did not differ significantly from those of the control. The analysis of the protein profiles of the purified myelin from the Shiverer showed a greatly decreased proportion of proteolipid protein, and almost complete absence of small and large basic proteins, and intermediate protein. Woldgram protein accounted for a much larger percentage of the total myelin protein than is the case in myelin from the control. Proteolipid protein, small and large basic proteins, and intermediate protein were found to be undetectable or decreased in various parts of the central nervous system from the Shiverer. Morphological observation by optic and electron microscope showed that the myelination of the optic nerve was equally affected as the spinal cord.

Evidence for the presence of L-arginine in the glial components of the peripheral nervous system.

L-Arginine is a precursor of nitric oxide that has been identified as an endogenous activator of soluble guanylate cyclase. We have recently reported the immunocytochemical localization of free L-arginine in glial cells in the central nervous system (CNS) using specific anti-arginine antibody. In the present study, we focused our attention on this particular amino acid in the peripheral nervous system (PNS). In the cochlea of the inner ear, arginine-like immunoreactivity was localized in satellite cells surrounding neurons of the spiral ganglion. In the dorsal root ganglia, satellite cells surrounding sensory neurons were found to be immunoreactive. In the superior cervical ganglion, L-arginine was concentrated in satellite cells around neuronal cells. In ganglia of the enteric plexus, supporting cells that covered neuronal cells were stained. These results show that free L-arginine in the PNS is concentrated in satellite and supporting cells, both of which correspond to glial cells in the CNS. Thus, those cells in ganglia of the PNS may support and/or control the neural activity by providing L-arginine to the neurons that they surround.

When does GABA-like immunoreactivity appear in the rat cerebellar GABAergic neurons?

The time of appearance of gamma-aminobutyric acid (GABA), a well-known neurotransmitter, during the development of cerebellar GABAergic neurons in rats was investigated immunocytochemically using purified anti-GABA antibody. Sprague-Dawley rats were used at embryonic days 15, 16, 18, 19 and 21, and postnatal days 0, 1, 5, 10, 15, 20 and 30. Golgi cells showed processes and GABA-like immunoreactivity at embryonic day 16 during migration. Purkinje cells were found immunoreactive at embryonic day 18, when they arrived at their destination. The reactivity of the basket cell was already apparent at postnatal day 5, and was thought to appear just after the end of migration. In all of the GABAergic neurons, GABA-like immunoreactivity was visible much earlier than the time of synapse formation and the emergence of their electrophysiological activity described in the literature. In addition, GABA-like immunoreactivity tended to shift from the soma and dendrite into the axon with development.

Predominant localization in glial cells of free L-arginine. Immunocytochemical evidence.

Nitric oxide has been recently identified as an endogenous activator of the soluble guanylate cyclase in the brain as well as in vascular endothelial cells and macrophages. In the present study, we determined the localization of free arginine in the brain because nitric oxide was formed from the terminal guanido group of L-arginine. Anti-arginine antiserum was raised in guinea pigs by repeated injection of L-arginine covalently conjugated to guinea pig serum albumin via glutaraldehyde. Specific anti-arginine antibody was purified from the antiserum by using an affinity gel coupled with L-arginine. Arginine-like immunoreactivity in the rat brain and spinal cord was found concentrated mainly in astrocytes including Bergmann glial cells in the cerebellum and processes of astrocytes around blood vessels. The present results suggest that glial cells, particularly astrocytes, are the main locus of L-arginine, a nitric oxide precursor, in the brain.

Biochemical and immunohistochemical studies on dysmyelination of quaking mutant mice in vivo and in vitro.

Dysmyelination in the central nervous system of the quaking mutant mouse was studied biochemically and immunohistochemically. We found, by measuring CNPase activity, that myelination in the central nervous system of quaking mice was affected to a different degree in different areas. The pallium cerebri was the most severely affected and the medulla and spinal cord were least affected. The density of astroglia observed by GFA staining wash higher in the white matter of quaking mice than in controls, but the total area of the white matter in the cerebellum was smaller in the quaking mice than in the controls. The DNA content in the pallium cerebri and brain stem showed no increase and that of the cerebellum was even lower in quaking mice than in the controls. Hypertrophy of the astroglia was observed in the white matter of the cerebellum of quaking mice, though Bergmann astroglial fibers in the molecular layer did not show any hypertrophy. The cerebella of quaking mice in primary culture showed very poor myelination under the phase-contrast microscope. However, Purkinje cells from the quaking mutants appeared normal with regard to Bodian silver impregnation, hematoxylineosin staining, and Purkinje cell specific P 400 protein. Addition of the conditioned culture medium of qk/qk to the culture of the control cerebellum did not interfere with the myelination. We concluded that the cause of dysmyelination in the quaking mouse could be a genetic defect of the oligodendroglia rather than hypertrophy of the astroglial cells.

Localization of nitric oxide-related substances in the peripheral nervous tissues.

Nitric oxide (NO) is now recognized as a transduction molecule in many biological systems, and is known to promote the synthesis of cGMP by activating the soluble guanylate cyclase. NO synthase which fully accounts for all the neuronal activity of NADPH diaphorase catalyzes L-arginine to NO and L-citrulline. In the present study, the localization of NO-related substances, L-arginine, NO synthase, L-citrulline and cGMP in the enteric plexus and dorsal root ganglia was demonstrated with immuno- or enzyme-histochemical methods. L-Arginine was proved accumulated in glial cells, while NO synthase and L-citrulline were found in neurons. Cyclic GMP was predominantly observed in glial cells. These results reveal L-arginine-NO-cGMP pathway may be present in the enteric plexus and dorsal root ganglion as in the brain, and provide visible evidence that NO mediates neuron-glia communications in this pathway.

Glycine-like immunoreactivity in the rat auditory pathway.

From neurophysiological and biochemical studies it has been suggested that glycine can function as a major inhibitory neurotransmitter in the central nervous system of mammals. In the present study, anti-glycine antiserum was obtained from rabbits immunized with glycine conjugated to rabbit serum albumin via glutaraldehyde and purified by affinity chromatography. The antibody thus obtained was found specific for glycine as determined by an enzyme immunoassay system. The immunocytochemical distribution of glycine in the auditory tract and internal ear was investigated with the antibody. In the central auditory pathway, glycine-like immunoreactivity was mainly located in the ventral and dorsal cochlear nuclei, trapezoid body, lateral lemniscus and inferior colliculus. In the labyrinth, immunoreactivity was detected in the vestibular ganglion and the supporting cells of the crista ampullaris and the organ of Corti, but not in the spiral ganglion. These findings suggest an important role of glycine in the auditory and vestibular pathways.

Coexistence of parvalbumin and glycine in the rat brainstem.

The coexistence of glycine- and PV-immunoreactivities was studied immunocytochemically in the nuclei of the superior olive, trapezoid body, cochlea and lateral lemniscus. All of the PV-immunoreactive neurons in the nuclei of the superior olive and trapezoid body were immunoreactive to glycine but not to GABA. In the dorsal cochlear nucleus, PV-positive neurons were sometimes immunoreactive to glycine. In the ventral nucleus of the lateral lemniscus, PV-positive cells were immunoreactive neither to glycine nor to GABA. Consequently, it was concluded that PV-immunoreactivity was distributed not only in the GABAergic neurons, but also in the glycinergic neurons and possibly in wider neuronal populations.