Use of Air-Liquid Two-Phase Flow in Hydrophobic Microfluidic Channels for Disposable Flow Cytometers

This paper describes a disposable flow cytometer that uses an air-liquid two-phase microfluidic system to produce a focused high-speed liquid sample stream of particles and cells. The susceptibility of thin liquid columns to instabilities may suggest that focusing of sample liquids with streams of air would be difficult. The design of channel geometry, control of flow rates, and use of appropriate surface chemistries on the channel walls, however, enabled the generation of thin (15–100 m) and partially bounded sample streams that were stable and suitable for rapid cell analysis. Using an inverted epi-fluorescence microscope with a photo-multiplier tube, we demonstrated that the system is capable of counting the number of beads and C₂C₁₂ myoblast cells. The effects of different flow rates and surface chemistries of the channel walls on the air-liquid two-phase flows were characterized using optical and confocal microscopy. Use of air instead of liquids as a sheath fluid eliminates the need for large sheath liquid reservoirs, and reduces the volume and weight requirements. The low manufacturing cost and high volumetric efficiency make the air-sheath flow cytometer attractive for use as a stand-alone device or as an integrated component of bio-artificial hybrid microsystems.     

FAMILIAL NEPHROPATHY ASSOCIATED WITH HEPATIC TYPE OF GLYCOGEN STORAGE DISEASE

The female patient was diagnosed as having Von Gierke's disease at 14 years of age, based on clinical manifestations, laboratory examination and liver biopsy. At 19 years of age she had uremia and died from its deterioration at 24 years of age. The parents were consanguineous, and a 27-year-old sister is presently hospitalized for renal Insufficiency with hepatomegaly. On autopsy, the patient's kidneys were highly contracted and contained a number of small cysts, mainly in the medulla. Histological examination indicated periglomerular fibrosis, glomerular hyalinization, tubular atrophy or cystic dilatation and intersitial fibrosis with round cell infiltration. These findings correspond to Fanconi's familial juvenile nephronophthlsis, except for age. The liver was markedly enlarged and Indicated severe glycogen deposits, but the kidney did not contain glycogen deposits. It can, therefore, be presumed that the renal lesions were not a secondary consequence of long-term glycogen deposits but that renal and hepatic lesions were associated with each other.     

A serratial protease causes vascular permeability reaction by activation of the Hageman factor-dependent pathway in guinea pigs.

The 56-kilodalton (56K) protease isolated from a culture filtrate of Serratia marcescens caused vascular permeability enhancement followed by edema formation when injected into guinea pig peripheral corneas and subconjunctival space or skin. The character and the mechanism of permeability enhancement were analyzed in vivo. The enhancement was maximum at 5 to 10 min. The permeability reaction increased exponentially by the amount of enzyme used. The enhancement of permeability induced by the 56K protease was not affected by treatment with an antihistamine but was greatly augmented by simultaneous injection of a kinin potentiator, Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro-OH (SQ 20,881). Furthermore, the permeability activity of the protease, but not the amidolytic activity, was inhibited by soybean trypsin inhibitor, a well-known inhibitor of plasma kallikrein, as well as by corn trypsin inhibitor, the best inhibitor of activated Hageman factor. Results of these in vivo studies indicate that the permeability-enhancing reaction induced by the 56K protease is caused by activation of the Hageman factor-dependent pathway in the tissue. The permeability-increasing activity of the 56K protease was parallel with the enzyme activity. Serratial lipopolysaccharide did not produce a permeability enhancement reaction within 30 min when injected into guinea pig skin. These results are consistent with the results of recent in vitro experiments in which activation of the purified Hageman factor but not of prekallikrein by the 56K protease was elucidated (Matsumoto et al., J. Biochem. (Tokyo) 96:739-749, 1984). Thus, the molecular mechanism described above appears to be operative in the pathogenesis of corneal edema and chemosis, which is induced by S. marcescens, in addition to the direct tissue destruction by the protease.     

Inhibitory effect of estradiol-17 beta and progesterone on bactericidal activity in uteri of rabbits infected with Escherichia coli.

The influence of ovarian hormones at different estrous stages on the bactericidal activity of the uterus in rabbits was investigated. When Escherichia coli cells were inoculated in ligated uteri, the survival period of the bacteria in the uterus at the luteal phase was clearly longer than that at the follicular phase. At the luteal phase, high levels of plasma estradiol-17 beta and progesterone were detected. A luteolytic treatment with prostaglandin F2 alpha and human chorionic gonadotropin at the luteal phase lowered plasma progesterone levels and prompted bacterial clearance from the uterus. In ovariectomized rabbits, E. coli from the uterine exudates was not detected 6 days after the inoculation in both the nontreated and estradiol-17 beta-treated animals. In the progesterone-treated rabbits, the survival period of E. coli was longer than that in the nontreated and estradiol-17 beta-treated animals. When estradiol-17 beta and progesterone at the ratio of 1:100 were administered concurrently, E. coli survived for the longest period in the rabbits treated with various doses of different hormones. Formalin-killed E. coli cells were inoculated into the uterine lumen, and 4 h later the proportion of heterophils phagocytizing the bacteria dropped in the progesterone-treated rabbits and in the estradiol-17 beta- and progesterone-treated rabbits, but there was no significant difference in heterophil numbers among the rabbits treated with different hormones. The present results suggest that progesterone inhibits the bactericidal activity of the uterus and that estrogen concurrently secreted at the luteal phase promotes the inhibitory action of progesterone, although estrogen alone hardly affects the uterine defense. In addition, the lowering of the bactericidal activity of the uterus at the luteal phase may be attributable to lower activity of phagocytosis by heterophils infiltrated into the uterine lumen.     

Cleavage of integrin by mu-calpain during hypoxia in human endometrial cells

PROBLEM: The distribution and activation of mu-calpain and possible cleavage of integrin in human endometrial cells under hypoxic condition were investigated. METHOD OF STUDY: Human endometrial epithelial and stromal cells were subjected to hypoxia, and subsequently used for immunostaining and western blot analysis. RESULTS: The proform of mu-calpain was detected in the cytoplasm of normal cells, and displayed a substantial decrease after hypoxia. Conversely, the active form of mu-calpain was not detected in normal cells, but was abundant after hypoxia. The cytoplasmic domain of integrin beta3 was also detected in the cytoplasm of endometrial cells. Western blot analysis confirmed that both the proform of mu-calpain and the integrin beta3 cytoplasmic domain decreased during hypoxia. CONCLUSIONS: Mu-calpain is activated in human endometrial cells during hypoxia and that subsequent cleavage of the integrin beta3 cytoplasmic domain may give some adverse effects to the function of human endometrium.     

Lung adenocarcinoma with bronchioloalveolar carcinoma component is frequently associated with foci of high-grade atypical adenomatous hyperplasia

We assessed the occurrence of atypical adenomatous hyperplasia (AAH) in whole lung lobes with primary cancer lesions. Following surgical resection, tissue specimens were sliced to a thickness of 4 mm (3,641 specimens from 61 cases; mean = 59.7 specimens per case). A total of 119 AAH foci were found and an association was evident in 25 (57%) of 44 adenocarcinomas, 3 (30%) of 10 squamous cell carcinomas, and 2 (29%) of 7 other lung cancers. Histologic evaluation showed that 108 AAH foci were categorized as low-grade and the other 11 as high-grade AAH. These 11 foci of high-grade AAH were present in 7 patients with adenocarcinoma, and in 1 patient there was a synchronous double primary lung adenocarcinoma. High-grade AAH was closely associated with bronchioloalveolar carcinoma (BAC) type adenocarcinoma, and low-grade AAH with non-BAC adenocarcinoma. The mean +/- SD Ki-67 labeling index in high-grade AAH (3.5%+/-2.9%) was significantly higher than for the low-grade index (1.4%+/-1.6%). We propose that foci of high- but not low-grade AAH may be potential precursor lesions of lung adenocarcinoma, especially with the BAC component.     

Role of apoptosis controlled by cytochrome c released from mitochondria for luteal function in human granulosa cells

PROBLEM: The aim of this study was to investigate the relationship between apoptosis by the mitochondrial pathway and luteal function in human granulosa cells. METHOD OF STUDY: Granulosa cells were obtained by ultrasound-guided follicular aspiration from patients undergoing in vitro fertilization and embryo transfer. After the addition of RU486, cells were stained with a mitochondria-specific fluorescent dye, MitoTracker Red CM x Ros. Using flow cytometry and National Institute of Health image, the mitochondrial fluorescent area was measured. After staining with Hoechst 33258 dye, the number of apoptotic bodies per 1000 cells were counted at random on photomicrographs. Homogenates were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis using antibodies against cytochrome c or caspase-3. RESULTS: The incidence of apoptotic bodies increased and the mitochondrial membrane potential decreased time dependently. The opposite effect was observed dose dependently with RU486 treatment. Western blot analysis showed increased cytochrome c expression, after treatment with 1-2 microg/mL of RU486 which then decreased with 5-10 microg/mL of RU486. Caspase-3 expression increased dose dependently with RU486. CONCLUSIONS: These results suggest that the activation of caspase-3 caused by cytochrome c released from mitochondria plays an important role in apoptosis-related luteal function in human granulosa cells.     

Disseminated Atypical Mycobacteriosis

Two cases of disseminated infection caused by Mycobacterium intracellulare were reported and discussed. In the first case, the patient was a fifty-seven-year-old male who complained of general fatigue, weight loss, and fever. Biopsy of the right inguinal lymph nodes and the liver revealed infiltration by histiocytes engulfing many acid-fast bacilli. At autopsy an egg-sized abscess was found is the region of the right Iliac lymph nodes. Histological examination showed histiocytic infiltration in the abscess wall, neighboring lymph nodes, liver, and spleen. In the second case, the patient was a four-year-old boy, who had persistent fever and splenomegaly. Splenectomy was performed and histological examination of the spleen revealed multiple nodular infiltration by swollen histiocytes with many acid-fast bacilli in their cytoplasm. The bone marrow aspirates and liver tissue obtained in the necropsy also showed many histiocytes containing many acid-fast bacilli. The authors emphasized the importance of paying special attention to atypical mycobacteriosis in feverish patients having lesions with a proliferation of histiocytes.     

Long-term inhibition of intimal hyperplasia using vascular photodynamic therapy in balloon-injured carotid arteries

Flexible treatments for intimal hyperplasia after angioplasty are still needed. The aim of this study was to demonstrate the long-term effects of vascular photodynamic therapy with talaporfin sodium on intimal hyperplasia following interventional injury. Intimal hyperplasia was induced by balloon distension injury to the carotid artery in 31 rabbits. Talaporfin, 5.0 mg/kg, was delivered systemically immediately after balloon injury. The injury site was irradiated with a diode laser light of wavelength 664 nm using a fluence of 50 J/cm² after 30 min. At day 3 and weeks 3, 6, 9, 15, and 25 after photodynamic therapy, the treated artery of each rabbit was excised and examined immunohistochemically. Thirty minutes after talaporfin administration, drug fluorescence was found only in the balloon-injured carotid artery wall. At 3 days, no smooth muscle cells were seen in the media of the photodynamic therapy-treated arterial segments. Intimal hyperplasia developed progressively in the balloon-injured and untreated segments; however, in the segments treated with photodynamic therapy, intimal hyperplasia was markedly suppressed until 25 weeks and the media was repopulated by smooth muscle cells without macrophages. Vascular photodynamic therapy with talaporfin may be used to inhibit restenosis after vascular intervention. An erratum to this article is available at .