Effects of subminimal inhibitory concentrations of minocycline on neutrophil chemotactic factor production in comedonal bacteria,neutrophil phagocytosis and oxygen metabolism

Summary Comedonal bacteria, Propionibacterium acnes, P. granulosum and coagulase-negative staphylococci (CNS) seem to play an important initiating role in the inflammatory process by producing neutrophil chemotactic factors. The attracted neutrophils, after phagocytosis, release inflammatory factors such as reactive oxygen species (ROS). We investigated the effects of minocycline at subminimal inhibitory concentrations (sub-MIC), i.e. one-tenth MIC, on the production of human neutrophil chemotactic factors in comedonal bacteria, and on several inflammatory parameters of neutrophils, including neutrophil phagocytosis and generation of ROS (O ₂ ^– , H₂O₂, O ). ROS generation in a cell-free, xanthinexanthine oxidase system was also assessed. Production of neutrophil chemotactic factors in all strains of P. acnes, P. granulosum and CNS were significantly suppressed by sub-MIC minocycline. Sub-MIC minocycline effectively reduced three kinds of neutrophil-generated ROS (O ₂ ^– , H₂O₂, O ). However, neutrophil phagocytosis and the ROS generated in a cell-free system were not markedly changed in the presence of sub-MIC minocycline. The results suggest that sub-MIC minocycline has an anti-inflammatory effect by inhibiting the production of neutrophil chemotactic factors in comedonal bacteria as well as ROS generated by neutrophils in the inflammatory process of acne.     

Suppressive effects of linoleic acid on neutrophil oxygen metabolism and phagocytosis

On the basis of recent reports that the proportion of linoleic acid (C18:2Cis 9,12), a free fatty acid, is markedly decreased in acne comedones and that tetracycline is effective against acne comedones by acting directly as an antioxidant on infiltrating neutrophils, we investigated the effect of linoleic acid on several inflammatory parameters of neutrophils, including neutrophil chemotaxis, phagocytosis, and generation of reactive oxygen species (ROS). Linoleic acid significantly decreased phagocytosis and the generation of O2-, H2O2, and OH.by neutrophils, whereas it did not significantly inhibit neutrophil chemotaxis or decrease the ROS levels generated in a cell-free, xanthine-xanthine oxidase system. The present study seems to suggest that decreased levels of linoleic acid in acne comedones contribute, in part, to the worsening of acne inflammation by the failure of low levels of linoleic acid to suppress neutrophil phagocytosis and ROS generation.     

Effect of palmitic acid on neutrophil functions in vitro

BACKGROUND: It has been shown that in acne comedones the proportion of linoleic acid is markedly decreased, while palmitic acid is significantly increased. We previously reported that the decreased proportion of linoleic acid, which markedly suppresses neutrophil reactive oxygen species (ROS) generation and phagocytosis, contribute to the worsening of acne inflammation. The aim of this study was to examine the effect of palmitic acid on neutrophil functions in vitro. METHODS: We investigated the effect of palmitic acid on inflammatory parameters such as neutrophil chemotaxis, phagocytosis, and ROS generation. Reactive oxygen species generation in a cell-free, xanthine-xanthine oxidase system was also assessed. The species examined were superoxide radical anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (OH.). RESULTS: Palmitic acid significantly decreased H2O2 generation both by neutrophils and in the xanthine-xanthine oxidase system, while neutrophil chemotaxis and phagocytosis as well as O2- and OH. generation by both systems were not markedly affected in the presence of palmitic acid. CONCLUSIONS: The present study suggests that palmitic acid may be involved in the pathogenesis of acne inflammation from a standpoint of oxidative tissue injury.     

A possible mechanism of action for azelaic acid in the human epidermis

Summary Azelaic acid, and other saturated dicarboxylic acids (C₉-C₁₂), are shown to be competitive inhibitors of tyrosinase (K _I azelaic acid = 2.73×10^–3 M) and of membrane-associated thioredoxin reductase (K _I azelaic acid = 1.25×10^–5 M). The monomethyl ester of azelaic acid does not inhibit thioredoxin reductase, but it does inhibit tyrosinase, although double the concentration is necessary compared with azelaic acid (K _I azelaic acid monomethyl ester = 5.24×10^–3 M). Neither azelaic acid nor its monomethyl ester inhibit tyrosinase when catechol is used as a substrate instead of l-tyrosine. Therefore, the weak inhibitory action of azelaic acid on tyrosinase appears to be due to the competition of a single carboxylate group on this inhibitor for the -carboxylate binding site of the l-tyrosine substrate on the enzyme active site. Based on the inhibitor constant on tyrosinase, at least cytotoxic levels of azelaic acid would be required for the direct inhibition of melanin biosynthesis in melanosomes if this mechanism is responsible for depigmentation in the hyperpigmentation disorders lentigo maligna and melasma. Alternatively only 10^–5 M azelaic acid is required to inhibit thioredoxin reductase. This enzyme is shown to regulate tyrosinase through a feedback mechanism involving electron transfer to intra-cellular thioredoxin, followed by a specific interaction between reduced thioredoxin and tyrosinase. Furthermore, the thioredoxin reductase/thioredoxin system is shown to be a principal electron donor for the ribonucleotide reductases which regulates DNA synthesis. Inhibition of thioredoxin reductase by azelaic acid provides a rationale for both its depigmenting property and the reversible inhibition of DNA synthesis observed in cultured epidermal cells and also in some of the bacteria associated with acne vulgaris.     

Effects of cepharanthin on neutrophil chemotaxis, phagocytosis, and reactive oxygen species generation.

The effects of cepharanthin on inflammatory parameters such as neutrophil chemotaxis, phagocytosis and reactive oxygen species (ROS) generation, were examined. Cepharanthin significantly decreased the levels of O2-, H2O2, and OH. generated by neutrophils. H2O2 and OH. generated in a cell-free, xanthine-xanthine oxidase system were also reduced in the presence of cepharanthin. However, the drug did not affect neutrophil chemotaxis or phagocytosis. The present study indicates that cepharanthin is an effective ROS scavenger, exerting its anti-inflammatory action by reducing the potent ROS species excessively generated in tissues and organs, especially at the sites of inflammation.     

Anti-oxidant effects of retinoids on inflammatory skin diseases

Summary It is well known that retinoids are effective in the treatment of various dermatological disorders. It has been reported that retinoids have inhibitory effects on the generation of superoxide (O ₂ ^- ) by stimulated polymorphonuclear leukocytes (PMNs). In the present study, we investigated the effects of retinoids on the generation of O ₂ ^- and other reactive oxygen species (ROS), including OH, H₂O₂ and chemiluminescence, by zymosan-stimulated PMNs and in the xanthinexanthine oxidase system, because these potent ROS may play an important role in PMN-mediated skin inflammation. It was found that some retinoids had anti-oxidant effects in the PMN system; however, apart from the effect of tretinoin and Ro 10-1670 on OH generation, none of the retinoids studied inhibited ROS generation in the xanthine-xanthine oxidase system. On the basis of these results, we discuss a possible mechanism connected with the role of ROS by which retinoids have favourable effects on several inflammatory skin disorders.     

Lysophosphatidylcholine: a chemoattractant to human T lymphocytes

Various cell stimuli act through activation of phospholipase A₂ resulting in the release of arachidonic acid, the precursor of eicosanoids, from the sn-2 position of cell membrane phospholipids. A byproduct of phospholipase A₂ activity is the lysophospholipids which have been found to potentiate T-lymphocyte activation. The purpose of the present study was to determine whether the various lysophospholipids modulate the migration of peripheral normal human T lymphocytes in vitro. It was found that lysophosphatidylcholine (lysoPC) induced T-lymphocyte migration in the concentration range 10^–7 to 10^–4 M with a maximum at 10^–6 M (mean chemotactic index, 2.06). The migration was due to chemotaxis rather than chemokinesis. In contrast, lysophosphatidylethanolamine (lysoPE) and lysophosphatidylinositol (lysoPI) did not exhibit chemotactic properties towards T lymphocytes. Further studies showed that the length of the fatty acids in the sn-1 position as well as the presence of double bonds modulated the chemotactic ability. The lysoPC compound with the highest chemotactic activity was lysoPC;1-palmitoyl (C=160). The results demonstrated that lysoPC, a phospholipase A₂-generated hydrolysis product of phosphatidylcholine, induced T-lymphocyte chemotaxis in vitro. Because phosphatidylcholine is the major phospholipid in the epidermis, the activation of phospholipase A₂ may result in the release of lysoPC in concentrations capable of inducing migration of T lymphocytes into the epidermis.     

Impaired neutrophil functions in patients with leukocytoclastic vasculitis

Background Leukocytoclastic vasculitis (LV) is characterized by segmental inflammation of small blood vessels, resulting in ischemic damage to the surrounding tissue. It is considered to be related to a type III hypersensitivity reaction, although the exact etiologic mechanism is not clear. Objective The purpose of this study was to evaluate neutrophil functions in patients with LV in order to understand their role in the pathogenesis of the disease. Methods Neutrophil functions were examined in 25 LV patients. The patients were divided into two groups: Group A consisted of 14 patients with drug-induced LV and Group B consisted of 11 patients where LV was induced by other factors. Results Both groups of patients showed significantly reduced chemotaxis and phagocytosis. Superoxide generation was significantly lower (P < 0.001) only in neutrophils from patients in Group A: 5.8 ± 0.5 nmoles O₂/10⁶ cells/min compared to 9.08 ± 0.8 nmoles O2/106 cells/min in the controls. Preincubation on normal neutrophils with the patients' sera caused an increase in their superoxide generation in accordance with the high IL-8 levels in these sera. Conclusions Neutrophil functions were significantly impaired in patients with LV. It is likely that factors present in LV plasma may chronically activate neutrophils, so that they become refracfory to further stimulation. Our study showed that neutrophil superoxide generation is low only in drug-induced LV; this test may assist in distinguishing such patients from those with LV induced by other causes.     

The inhibition of free radical generation by human neutrophils through the synergistic effects of metronidazole with palmitoleic acid: a possible mechanism of action of metronidazole in rosacea and acne

Summary Metronidazole is clinically effective in treating not only rosacea but also acne inflammation. Yet it is generally considered not to be very effective in inhibiting the growth of anaerobic Propionibacterium acnes. We report here our investigation into the synergistic effects of metronidazole and palmitoleic acid on the anaerobic growth of P. acnes as well as on human neutrophil functions, including the generation of reactive oxygen species (ROS). Both metronidazole and palmitoleic acid, when used alone, only slightly inhibited the growth of P. acnes, and no significant decrease in human neutrophil functions, including the generation of ROS, was observed. But metronidazole used in the presence of palmitoleic acid markedly inhibited the anaerobic growth of P. acnes and decreased ROS generation by neutrophils. However, ROS generated in the xanthine-xanthine oxidase system were not affected. Metronidazole was shown to be clinically effective by decreasing neutrophil-generated ROS at the sites of inflammation with the aid of palmitoleic acid, which is generally present in human skin. By inhibiting oxidative tissue injury under in vivo conditions, treatment with metronidazole results in remarkable improvement of rosacea and acne.     

Increased in vivo secretory activity of neutrophil granulocytes in patients with psoriasis and palmoplantar pustulosis

Summary The relationship between psoriasis and palmoplantar pustulosis (PPP) is uncertain, as is the role of the neutrophil granulocyte in these conditions. In a previous comparative study of the rate of polymorphonuclear leucocyte (PMN) phagocytosis of IGG- and IgG-C3b-coated particles, an increased uptake rate was found in both diseases. Further information on the in vivo activity of PMNs in these conditions may be obtainable by determining the level of lactoferrin (LF) in serum from such patients, since LF serves as a specific marker of the turnover and activity of the circulating pool of neutrophils. In this study on 19 patients with psoriasis and 20 patients with PPP, elevated levels of LF were found in both conditions. In contrast, the levels of lysozyme and ₂-microglobulin, which are markers of monocyte-macrophage and lymphocyte activity, respectively, were normal. This suggests the selective activation of neutrophils in these disorders. LF was significantly correlated (P<0.05 and 0.001, respectively) to the rates of phagocytosis of IgG- and IgG-C3b-coated particles, but not to the chemotaxis of isolated PMNs. There was no correlation between the severity of the disease and the levels of serum LF. The data suggest the increased in vivo activity of neutrophils in psoriasis and PPP.     

Comparison of Azelaic Acid and Anthralin for the Therapy of Patchy Alopecia Areata

Background: Although topical azelaic acid has been previously used for the treatment of alopecia, no controlled trials of azelaic acid for this condition have been conducted to date. Objective: The goal of this study was to determine the efficacy, tolerability, and safety of azelaic acid treatment in patients with patchy alopecia areata (AA) in comparison with anthralin (dithranol) treatment. Subjects and methods: This study included 31 subjects with patchy AA who did not receive any treatment for at least 1 month prior to the study. Demographic and clinical characteristics of these subjects were recorded at baseline. Subjects were randomized to apply either 20% azelaic acid (15 subjects) or 0.5% anthralin (16 subjects) for 12 consecutive weeks. In a subsequent 8-week follow-up period no cream was applied. Two independent investigators performed an efficacy evaluation with clinical examination using a terminal hair regrowth score (RGS) with a scale ranging from 0 (inadequate response) to 2 (complete response) at week 20. Partial response was accepted as score 1. Results: Both groups were well matched for the relevant demographic and clinical indicators affecting treatment response at baseline. All subjects completed the trial. At week 20 the RGS was 1.27 ± 0.9 in the azelaic acid group versus 1.37 ± 0.8 in the anthralin group (p > 0.05). A complete response was observed in 53.3% of cases in the azelaic acid group (8 of 15) compared with 56.2% (9 of 16) in the anthralin group (p > 0.05). No serious adverse events were observed in either group during the study. Conclusion: The present pilot study showed that the use of azelaic acid gave similar results to anthralin with regard to hair regrowth, and that it can be an effective topical therapy for patchy AA. More extensive trials are necessary, however, to reach a definitive conclusion.     

Calcipotriol (MC 903), a novel vitamin D3 analogue stimulates terminal differentiation and inhibits proliferation of cultured human keratinocytes

Summary The hormonally active form of vitamin D₃, 1,25-dihydroxy vitamin D₃ [1,25-(OH)₂-D₃; calcitriol], regulates the differentiation and proliferation of epidermal keratinocytes in vitro. MC 903 (calcipotriol) is a novel vitamin D₃ analogue which is at least 100 times less potent than 1,25-(OH)₂-D₃ in its effects on calcium homeostasis. The present study compared the effects of MC 903 and 1,25-(OH)₂-D₃ on terminal differentiation and proliferation of cultured normal human keratinocytes. Keratinocytes were grown in McCoy's 5A medium supplemented with penicillin (50 IU/ml), streptomycin (50 g/ml), l-serine (4×10^–4 M), and 10% human type AB serum. MC 903, 1,25-(OH)₂-D₃ or 1-OH-D₃ (10^–12 M–10^–8 M) was added with each feeding when cultures became preconfluent. After incubation for 24 h with D₃ vitamins, cultures were extracted for transglutaminase, and the enzyme activity was indexed against DNA content. The activity of transglutaminase, the enzyme reponsible for cross-linking the proteins of the cornified envelope, was maximally stimulated by 388% with MC 903 (10^–8 M), by 328% with 1,25-(OH)₂-D₃ (10^–8 M), and by 27% with 1-OH-D₃ (10^–8 M) compared with vehicle. After incubation for 2 weeks the number of keratinocytes with cornified envelopes had increased by 288% with MC 903 (10^–8 M), by 360% with 1,25-(OH)₂-D₃ (10^–8 M), and by 149% with 1-OH-D₃ (10^–8 M) compared with vehicle. Simultaneously the incorporation of (³H)thymidine into DNA was decreased by 64% with MC 903 (10^–8 M), by 71% with 1,25-(OH)²-D₃ (10^–8 M), and by 10% with 1-OH-D₃ (10^–8 M). There was a corresponding decrease in cell number. These results demonstrate that both MC 903 and 1,25-(OH)₂-D₃ are potent modulators of keratinocytes differentiation and proliferation in vitro. Because MC 903 is much less active than 1,25-(OH)₂-D₃ in causing hypercalcemia, this compound is a candidate for the treatment of skin diseases characterized by aberrant epidermal differentiation and proliferation.Parts of this study were presented at the ESDR meeting in Munich, May, 1988     

SUCCESSFUL TREATMENT OF A PATIENT WITH RECURRENT FURUNCULOSIS BY VITAMIN C: IMPROVEMENT OF CLINICAL COURSE AND OF IMPAIRED NEUTROPHIL FUNCTIONS

Background. Neutrophils play a critical role in host defense against a variety of microbial pathogens. There is much information to suggest a role for vitamin C in the physiology of neutrophils. Thus, the effects of vitamin C treatment were studied in a patient with a history of recurrent furunculosis who showed altered neutrophil functions. Methods. Superoxide generation was measured by cytochrome C reduction. Phagocytosis of opsonized zymosan by neutrophils and chemotaxis on agarose plates were determined. Results. Chemotaxis, phagocytosis, and superoxide generation of the patient's neutrophils were significantly lower than those of the matched control. Treatment with vitamin C (500 mg/day) for 30 days caused a dramatic clinical response and a significant improvement of all three neutrophil functions to values similar to those of the controls. Conclusions. We suggest that the patient described here had a temporary defect in neutrophil functions. The treatment with vitamin C probably prevented neutrophil oxidation, thus contributing to recovery of neutrophil function and arrest of furunculosis.     

Studies of polymorphonuclear migration into psoriatic skin using a new in vivo method

Summary A new method, employing a skin-implanted cell trap already used to study chemotaxis in cancer patients, was applied to 35 healthy volumteers and 12 psoriatic patients. A dacron disk impregnated with 10 l of 4–6·10⁶ live BCG suspension was implanted in the deep dermis. After 24 h the disk was removed, and five sections of each disk were counted for polymorphonuclear leukocytes (PMNs) and monocytes. Involved and uninvolved psoriatic skin showed a decrease of PMN migration into the disk as compared with controls. No difference could be demonstrated between involved and uninvolved skin. Mononuclear cell chemotaxis was the same in psoriasis as in controls. These results are in agreement with other in vivo data using mainly the skin chamber technique indicating a decrease of PMN chemotaxis in psoriatic skin at 24 h.     

Extensive xanthelasma associated with anaplastic large cell lymphoma and hyperimmunoglobulin E syndrome

A 57‐year‐old woman presented with a 6‐month history of an extensively spreading, yellowish patch on the periorbital areas and cheeks. A diagnosis of hyperimmunoglobulin E syndrome had been made at the age of 22 years on the basis of an eczematous eruption, recurrent furunculosis, and a persistently elevated immunoglobulin E (IgE) level. Her past medical history revealed that she had suffered from numerous recurrent bouts of chronic sinusitis, otitis media, oral candidiasis, orbital cellulitis, acne rosacea, and pneumonia caused by cytomegalovirus since her twenties. In addition, 1 year ago, anaplastic large cell lymphoma of the cervical lymph node (stage IIIb) developed, and she received six cycles of cyclophosphamide–doxorubicin–vincristine–prednisolone (CHOP) chemotherapy with partial remission. None of her family had any of these problems. Cutaneous examination showed extensive, symmetric, noninfiltrated macular areas of distinct yellow discoloration around the eyes and on both cheeks ( Fig. 1 ). There were also erythematous papulonodular eruptions on the nose and both cheeks, which were thought to be acne rosacea. Laboratory findings were normal, except for an elevated IgE level (8157 IU/mL). Serum concentrations of IgG, IgA, and IgM were normal. Serum complement levels were normal, as evidenced by normal C₃, C₄, and CH₅₀. Although she had a previous history of a decreased level (12%) of nitroblue tetrazolium (NBT) test (control, 53%), NBT test at our institute was normal. Neutrophil function tests, including neutrophil chemotaxis, neutrophil phagocytosis, neutrophil respiratory burst, and neutrophil microbial killing test, by flow cytometry, showed normal results. The serum lipid levels, including total cholesterol, triglyceride, low‐density lipoprotein‐cholesterol, and high‐density lipoprotein‐cholesterol, were normal. Serum lipoprotein electrophoresis was normal. A biopsy specimen revealed scattered foamy cells throughout the dermis. The larger clusters of foamy cells tended to group around the blood vessels of the dermis ( Fig. 2 ).

Figure 1 Open in figure viewer PowerPoint Extensively distributed, yellowish, flat xanthelasma on the face     

Zum Einfluß anorganischen Arsens auf die DNS-Synthese menschlicher Lymphocyten in vitro

Zusammenfassung An Phytohemagglutinin-stimulierten menschlichen Lymphocytenkulturen wurde die cytotoxische bzw. mutagene Wirkung von anorganischem Arsen in vitro geprüft. Den einzelnen Kulturansätzen wurde Na₂HAsO₄ in Konzentrationen zwischen 3 10^–5 g und 5 10^–8 g pro Milliliter Kulturmedium zugeführt. Als Kontrollen dienten gleichzeitig angelegte Parallelkulturen derselben Probantenohne Arsenzusatz.Wie unsere Untersuchungen zeigen, treten bereits bei relativ niederen den Kulturen zugefügten Arsendosen geringe bis schwere Kernteilungsstörungen auf. Beieiner Konzentration von 0,1 Na₂HAsO₄ pro Milliliter Kulturmedium sind 36,3% und bei mehr als 2,0 80–100% sämtlicher Metaphasenplatten pulverisiert.Auffallenderweise sinkt der Mitose-Index erst bei höheren Arsenkonzentrationen deutlich ab, während der ³H-Thymidin-Markierungs-Index gegenüber den Kontrollkulturen bereits bei den mittleren der von uns gewählten Arsendosen signifikant erniedrigt gefunden wird.Es werden folgende Schlußfolgerungen gezogen: Arsen kann den Phosphoreinbau in die Nucleotidketten kompetetiv hemmen. Ferner besitzt das Arsen eine inhibitorische Wirkung auf Sulfhydryl-haltige Enzyme, bzw. auf die Dark-Repair-Mechanismen. Infolge dessen kommt es zu einer Alteration des Zellstoffwechsels im Sinne einer Fehlbildung von DNS bzw. RNS, wobei der Einbau von³H-Thymidin in die DNS nicht wesentlich herabgesetzt zu sein braucht.Während der Dauer der Arsenexposition ist die Zelle nicht in der Lage, die dann an den Chromosomen gesetzten Läsionen zu erkennen und zu ersetzen. Es kommt zum Persistieren derselben, die dann als somatische Punktmutationen Ausgangspunkte von evtl. atypischen Zellpopulationen bilden können.Obwohl die vorliegenden Befunde an Lymphocyten erhoben wurden, ist anzunehmen, daß auch Zellen anderer Organsysteme (z.B. Epidermis) entsprechend alteriert werden können.

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The effect of inorganic arsenic on DNA-synthesis of human lymphocytes in vitro

Summary The cytotoxic and mutagenic effects of inorganic arsenic were tested in vitro on lymphocyte cultures stimulated by phytohemagglutinin. Na₂HAsO₄ in concentrations between 3 × 10^–5 g and 5 × 10^–8 g per ml of culture medium was added to the various cultures. Parallel cultures of the same test objects without addition of arsenic were used as controls.Slight to heavy disturbances of nuclear division appeared already in the presence of relatively low doses of arsenic added to the cultures. With a concentration of 1 × 10^–7 g Na₂HAsO₄ per ml culture medium 36,3%—and with more than 2 × 10^–6 g, 80–100%—of the entire metaphase plates were pulverized.The mitosis index decreases considerably only with higher arsenic concentrations, whereas the ³H-Thymidine labelling index in relation to the control cultures is found significantly diminished already at medium doses of arsenic.The following conclusions have been drawn:Arsenic can competitively inhibit the insertion of phosphorus into the nucleotide chains. Furthermore, arsenic has an inhibiting effect on enzymes containing sulfhydryl, or on the Dark Repair Mechanisms, respectively. Consequently, the cellular metabolism is altered towards a false formation of DNA and RNA, in which case the incorporation of³H-thymidine into DNA does not have to be reduced.During the time of exposition to arsenic the cell is not able to recognize and rebuild the lesions within the chromosomes, which then begin to persist and may form—as somatic point mutations—starting points for possible atypical cell populations.Although the present findings were achieved with lymphocytes it can be assumed that cells of different organic systems (e.g. epidermis), too, can be altered correspondingly.

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Die vorliegenden Untersuchungen wurden mit Unterstützung der Stiftung Volkswagenwerk, Hannover, durchgeführt.

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Wesentliche Teile dieser Arbeit wurden auf der Tagung der südwestdeutschen Dermatologen (Frankfurt am Main 4.–5. 6. 1971) vorgetragen.     

Effects of ethanol and acetaldehyde on phagocytic functions

Summary Although a number of skin diseases are characterized by the presence of an increased number of phagocytes in their lesions, the effects of alcohol on phagocytic functions are not clearly understood. Therefore, we measured the influence of ethanol and acetaldehyde on the generation of oxygen radicals, chemotaxis and the release of lysosomal enzymes from human phagocytes. We added 0.03%–3% ethanol and 0.005%–0.25% acetaldehyde to cell cultures. We found that both ethanol and acetaldehyde suppressed the generation of oxygen radicals from granulocytes and monocytes; the ID₅₀ was achieved at concentrations of approximately 0.25% for ethanol and 0.03% for acetaldehyde. A significant inhibition of granulocyte chemotaxis was first noted with 0.063% ethanol and 0.016% acetaldehyde. Ethanol and acetaldehyde inhibited the release of the lysozyme of monocytes at concentrations of >0.75% and >0.03% respectively, but granulocytes were unaffected; the release of -glucuronidase and lactate dehydrogenase remained stable. Due to the high volatility of the agents, especially acetaldehyde, under the experimental procedures employed, the actual concentrations of the agents were probably lower and similar to those measured in vivo. Our results indicate that defined phagocytic functions are strongly inhibited by concentrations of ethanol and acetaldehyde which are associated with moderate to severe inebriation.In partial fulfillment of an M. D. thesis     

Veränderungen der nucleinsäure-synthese durch arsen bei isolierten menschlichen lymphocyten unter anwendung der sedimentations-und ficoll-methode zur zellgewinnung

Zusammenfassung Der Grad der Na₂HAsO₄-bedingten Hemmung der Nucleinsäure-Synthese kultivierter menschlicher Lymphocyten ist abhängig von der Methode der Lymphocytengewinnung. Durch radioaktive Einbauversuche wurde nachgewiesen, daß—bei identischen Arsenkonzentrationen von über 10 µg/ml Kulturmedium — die Inkorporation von¹⁴C-Thymidin in die DNA von Lymphocyten, die mit der Ficoll-Methode isoliert wurden, signifikant höher lag, als in den Versuchsansätzen, deren Zellen mittels der Sedimentationsmethode gewonnen wurden. Eine Vorbehandlung von Ficoll mit Dow-Chelating Resin (Deionisation) bewirkt eine weitgehende Befundangleichung zwischen Ficoll- und Sedimentationsmethode. Handelsübliches nicht deionisiertes Ficoll verstärkt den mitogenen Effekt von PHA auf isolierte menschliche Lymphocyten um den Faktor zwei.Durch emissionsspektrographische Untersuchungen und Leitfähigkeitsmessungen konnte gezeigt werden, daß die sich unterscheidenden Befunde wahrscheinlich Folge eines Ionendefizits im handelsüblichen Ficoll sind. Dieses verursacht über eine Veränderung der Membranpotentiale eine Erhöhung der Zellpermeabilität mit Freisetzung Arsen-bindender Zellbestandteile aus den Lymphocyten. Die wirksame Arsenkonzentration im Inkubationsansatz kann möglicherweise zusätzlich vermindert werden durch eine direkte Adsorption von Arsenat durch noch vorhandene Ficoll-Reste in den Kulturansätzen.Der Einbau von¹⁴C-Uridin in die RNA von Lymphocyten, die mit handelsüblichem Ficoll gewonnen wurden, lag zwar auch über der Einbaurate jener Kulturen, die mittels der Sedimentationsmethode gewonnen wurden, jedoch konnte. dieser Effekt statistisch nicht abgesichert werden.

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Arsenic-dependent changes in nucleic acid synthesis in human lymphocytes obtained by sedimentation or ficoll gradient

Summary The degree of inhibition of the nucleic acid synthesis of cultivated human lymphocytes caused by Na₂HAsO₄ is dependent on the method of obtaining the lymphocytes. It was shown by means of radioactive incorporation experiments that—with identical arsenic concentrations of over 10µg/ml for the culture medium — the incorporation of¹⁴C-Thymidine into DNA of lymphocytes isolated by the Ficoll method was significantly higher than in the experimental batches obtained by means of the sedimentation method. A preliminary treatment of Ficoll with Dow Chelating Resin (deionization) causes an extensive equation of the results of the Ficoll and the sedimentation method. Commercial non-deionized Ficoll intensifies the mitogenetic effect of PHA on isolated human lymphocytes by approximately the factor two.It could be demonstrated by means of emission spectrographic analysis and conductivity measurements that the differing results are probably due to an ion deficit in the commercial Ficoll. This caused and increase in the cell permeability through a change of the membrane potentials, with the release of arsenic-binding cell components from the lymphocytes. The effective arsenic concentration in the incubation batch can possibly be additionally reduced by means of a direct adsorption of arsenate through Ficoll residues still present in the culture batches.The incorporation of¹⁴C-uridine into the RNA of lymphocytes obtained by commercial Ficoll was higher than the incorporation ratio of cultures obtained by means of the sedimentation method. However this effect could not be proved statistically.

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Herrn Prof. Dr. K. W. Kalkoff zum 65. Geburtstag gewidmet.

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Die Untersuchungen wurden mit Unterstützung der Stiftung Volkswagenwerk, Hannover, und der Deutschen Forschungsgemeinschaft, Bonn-Bad Godesberg, durchgeführt.     

Partial purification and characterization of an alkaline dipeptide naphthylamidase (Arg-Arg-NAase) of the rat skin

Summary A peptidase capable of cleaving N-terminal dipeptides from arginyl-arginine naphthylamide (Arg-Arg-NA), leucyl-alanine naphthylamide as well as from alanyl-alanyl-alanyl-alanine (but not alanyl-alanyl-alanine) was partially purified from rat skin homogenate by employing chromatography on DEAE-cellulose, gel filtration on Sephadex G-100 and DEAE-Sephadex. The final preparation was free from peptidases hydrolysing monoamino acid naphthylamides. The enzyme was optimally active at pH 8.0. The enzymic hydrolysis of Arg-Arg-NA was enhanced by chloride ions and sulfhydryl compounds. K _m value was 1×10^–4 M with Arg-Arg-NA as substrate.

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Zusammenfassung Eine Peptidase, die N-terminale Dipeptide von Arginyl-Arginin-Naphthylamid, Leucyl-Alanin-Napthhylamid und auch von Alanyl-Alanyl-Alanyl-Alanin (aber nicht von Alanyl-Alanyl-Alanin) liberieren kann, wurde von Rattenhaut durch Chromatographie an DEAE-Cellulose, Gel-Filtration an Sephadex G-100 und DEAE-Sephadex purifiziert. Das Enzympräparat war frei von Peptidasen, die Aminosäurenaphthylamide hydrolysieren. Die enzymatische Hydrolyse von Arginyl-Arginin-Naphthylamid war optimal bei pH 8,0 und wurde in Gegenwart von Chlorid-Ionen und Sulfhydryl-Gruppen gesteigert. Der K _m -Wert für die Hydrolyse von Arginyl-Arginin-Naphthylamid war 1 · 10^–4 M.

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This report forms part IV in our series on Peptidases in the skin, and is a part of the research project of the Skin Biology Research Unit in Turku (SBRU). Financial support was obtained as grants from the Sigrid Juselius Foundation and from the Finnish Medical Council to the senior author (V.K.H-H.).     

Organ culture of human hair follicles in serum-free medium

Human hair follicles were cultured in serum-free media at 31C in an atmosphere containing 95% O₂ and 5% CO₂. Results showed that the length of the cultured hair increased time dependently for 96 h. Histological findings revealed that the hair germinative cells maintained their normal morphology throughout the 96 h culture period. DNA synthesis in the hair bulb also increased time dependently for 96 h. Autoradiographs of ³H-thymidinelabelled follicles indicated that they were localized in the germinative cells below Auber's critical line. The effects of minoxidil sulphate on DNA synthesis in this culture system were concentration dependent. Minoxidil sulphate at concentrations of 10^–10,10^–9 and 10^–8 M significantly increased DNA synthesis compared with DNA synthesis in the control medium. Autoradiographs of the follicles cultured in 10^–10 M minoxidil sulphate showed that ³H-thymidine localized primarily in the germinative cells below Auber's critical line. These results suggest that this organ culture system may be useful for studying DNA synthesis by hair germinative cells in serum-free media.