Clofazimine, a Promising Drug for the Treatment of Babesia microti Infection in Severely Immunocompromised Hosts.

BACKGROUND: Persistent and relapsing babesiosis caused by Babesia microti often occurs in immunocompromised patients, and has been associated with resistance to antimicrobial agents such as atovaquone. Given the rising incidence of babesiosis in the United States, novel drugs are urgently needed. In the current study, we tested whether clofazimine (CFZ), an antibiotic used to treat leprosy and drug-resistant tuberculosis, is effective against B. microti. METHODS: Mice with severe combined immunodeficiency were infected with 107B. microti-infected erythrocytes. Parasites were detected by means of microscopic examination of Giemsa-stained blood smears or nested polymerase chain reaction. CFZ was administered orally. RESULTS: Uninterrupted monotherapy with CFZ curtailed the rise of parasitemia and achieved radical cure. B. microti parasites and B. microti DNA were cleared by days 10 and 50 of therapy, respectively. A 7-day administration of CFZ delayed the rise of parasitemia by 22 days. This rise was caused by B. microti isolates that did not carry mutations in the cytochrome b gene. Accordingly, a 14-day administration of CFZ was sufficient to resolve high-grade parasitemia caused by atovaquone-resistant B. microti parasites. CONCLUSIONS: Clofazimine is effective against B. microti infection in the immunocompromised host. Additional preclinical studies are required to identify the minimal dose and dosage of CFZ for babesiosis.

Experimental validation of biocompatible nanostructured lipid carriers of sophorolipid: Optimization, characterization and in-vitro evaluation.

To date, the potential of sophorolipids (an important class of glycolipids) has been exploited solely as amphipathic molecules but their ability to formulate lipid nanoparticles has never been explored. In this report, for the first time, lipid nanostructures coated with polysorbates (Tweens) were formulated by a hot dispersion method. By varying the amount of lipid, type of surfactant, and alcohol, dilution ratio etc., the formulation was optimized with respect to its stability, which is a central aspect of their potential applications. Their comprehensive physicochemical characterization was done using static and dynamic light scattering (SLS, DLS), small angle neutron scattering (SANS), zeta-potential, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) techniques. Further hemolysis study was conducted to understand the in-vitro cytotoxicity levels of the lipidic nanoparticles prior to its application as a potent drug delivery device for countermanding the problems associated with challenging tuberculosis and leprosy drug-Rifampicin. Attaining high entrapment efficiency and sustained release from the developed carrier, further interaction with bovine serum albumin was investigated, to understand the in-vivo behavior of the nanostructured lipid carriers (NLCs).

A study of the free radical scavenging effects of Piper betle leaf extract in patients with vitiligo.

BACKGROUND: Vitiligo is an idiopathic skin disease manifested by depigmented macules. It is characterised by melanocyte destruction, and redox imbalance is proposed to play a contributory role. AIM: The aim of this study was to analyze the effects of an ethanolic extract of Piper betle leaves on the generation of reactive oxygen species in erythrocytes sourced from vitiligo patients. METHODS: The effect of Piper betle on the generation of reactive oxygen species in erythrocytes was measured by flow cytometry in patients with active and stable vitiligo versus healthy controls, using 5-(and-6)-chloromethyl-2'-7'-dichlorodihydrofluorescein diacetate. RESULTS: The generation of reactive oxygen species in erythrocytes was higher in patients with vitiligo (n = 23) compared to healthy controls (n = 18). The geometrical mean fluorescence channel was 23.05 ± 2.11 in patients versus 17.77 ± 1.79 in controls, P = 0.039. The levels of reactive oxygen species were higher in patients with active vitiligo. Treatment of erythrocytes with Piper betle in concentrations of 0.5 and 1.0 µg/ml significantly decreased the baseline levels of reactive oxygen species by 31.7% in healthy controls, and 47.6% and 44.3% in patients with active vitiligo, respectively. Piper betle effectively scavenged hydrogen peroxide, which was evident by a decrease in the geometrical mean fluorescence channel by 52.4% and 62.9% in healthy controls, and 45.0% and 57.0% in patients with active vitiligo. LIMITATIONS: The study had a small sample size. Future studies should focus on evaluation of the antioxidant role of Piper betle at the lesional site. CONCLUSION: This pilot study indicates that patients with active vitiligo demonstrate enhanced generation of reactive oxygen species in erythrocytes, which was significantly reduced following ex vivo treatment with Piper betle.

Clofazimine Inhibits the Growth of Babesia and Theileria Parasites In Vitro and In Vivo.

The present study evaluated the growth-inhibitory effects of clofazimine, currently used for treating leprosy, against Babesia bovis, B. bigemina, B. caballi, and Theileria equi in in vitro culture and against Babesia microti in mice. The 50% inhibitory concentrations (IC50s) of clofazimine against the in vitro growth of B. bovis, B. bigemina, B. caballi, and T. equi were 4.5, 3, 4.3, and 0.29 µM, respectively. In mice infected with B. microti, treatment with 20 mg/kg of body weight of clofazimine administered orally resulted in a significantly lower peak parasitemia (5.3%) than that in the control group (45.9%), which was comparable to the subcutaneous administration of 25 mg/kg diminazene aceturate, the most widely used treatment for animal piroplasmosis. Although slight anemia was observed in both clofazimine- and diminazene aceturate-treated infected mice, the level and duration of anemia were lower and shorter, respectively, than those in untreated infected mice. Using blood transfusions and PCR, we also examined whether clofazimine completely killed B. microti On day 40 postinfection, when blood analysis was performed, parasites were not found in blood smears; however, the DNA of B. microti was detected in the blood of clofazimine-treated animals and in several tissues of clofazimine- and diminazene aceturate-treated mice by PCR. The growth of parasites was observed in mice after blood transfusions from clofazimine-treated mice. In conclusion, clofazimine showed excellent inhibitory effects against Babesia and Theileria in vitro and in vivo, and further study on clofazimine is required for the future development of a novel chemotherapy with high efficacy and safety against animal piroplasmosis and, possibly, human babesiosis.

Anti-inflammatory activity of superoxide dismutase obtained from Debaryomyces hansenii on type II collagen induced arthritis in rats.

INTRODUCTION: Rheumatoid arthritis is an autoimmune inflammatory disease of unknown etiology, free radicals have been implicated in the genesis and perpetuation of damage in this pathology. OBJECTIVE: To evaluate the anti-inflammatory effect of Cu,Zn-superoxide dismutase (SOD) obtained from two different sources (bovine erythrocytes, Be-SOD, and Debaryomyces hansenii, Dh-SOD) with Type II Collagen-induced Arthritis model in rats. MATERIAL AND METHODS: Arthritis was induced by repeated injection of a porcine type II collagen-incomplete Freund adjuvant suspension on the back of Dark Augui (DA) rats. Arthritis was clinically evaluated throughout the study. Body weight was determined at three different times. Two different doses for each treatment (Be-SOD, Dh-SOD) were tested: 100 and 1,000 U/kg. At the end of the trial (day 28), histological analyses of the most inflamed ankle joint, as well as serum anti-collagen antibodies, were determined. RESULTS: Both sources of SOD decreased, although to a different extent, the incidence and severity of the disease. Arthritis score was lower in all treatments, except for the low dose of Be-SOD. Groups receiving either source of SOD showed a significant weight increase compared to the placebo group. Histological damage was similar in all groups. Only the group that received the highest dose of Dh-SOD showed a significant lower antibody titer; nevertheless, no correlation appears to derive from arthritis score and antibody titer. CONCLUSION: Our findings suggest that, although unable to counteract the arthritis syndrome, SOD may still be beneficial due to its anti-inflammatory activity. In the case of Dh-SOD, the best effect was observed at the highest dose tested.

Erythrocyte glutathione peroxidase, glutathione reductase activities and blood glutathione content in leprosy.

OBJECTIVES: Leprosy is a chronic granulomatous infection caused by Mycobacterium leprae involving cutaneous tissue and peripheral nerves producing skin lesions, nerve degeneration, anaesthesia and deformities. In leprosy, the activated phagocytes produce reactive oxygen species (ROS) as a part of their microbicidal function. Such ROS are capable of damaging the host tissue by lipid peroxidation. Increased lipid peroxidation has been reported in leprosy. The glutathione antioxidant system with glutathione peroxidase (GSH-Px), glutathione reductase (GR) and glutathione (GSH) as components protect the cells from ROS toxicity and lipid peroxidation. The objective of the present study was to assess blood glutathione content and erythrocyte antioxidant enzyme activities of glutathione peroxidase and glutathione reductase in leprosy. DESIGN: The parameters were studied in 100 leprosy patients and 50 normal healthy controls. The data was analysed by grouping the patients into Ridley-Jopling (RJ) types [tuberculoid leprosy (TT), borderline tuberculoid leprosy (BT), borderline leprosy (BB), borderline lepromatous leprosy (BL), lepromatous leprosy (LL)] and into different levels of Bacteriological Index (BI) [bacteriologically negative (BI=0), BI=0.1-1, BI=1.1-2, BI=2.1-3, BI=3.1-4, BI=4.1-6]. METHODS: Venous blood sample was used for the study. The GSH level was estimated in the blood by DTNB [5,5'-dithiobis(2-nitrobenzoic acid)] reduction method. The enzyme activities were measured in the red blood cell haemolysate by kinetic methods using NADPH. RESULTS: GSH, GSH-Px and GR were significantly low in leprosy (total patients) as compared to the control group (p<0.001). A progressive decrease in GSH level and enzyme activities was noted along the leprosy spectrum from TT to LL. A significant decline of GSH in BB (p<0.05), BL (p<0.005) and LL (p<0.001); and of GSH-Px and GR in BT (p<0.05, p<0.02), BB (p<0.02), BL (p<0.005) and LL (p<0.001) was noted as compared to controls. A significant lowering of GSH-Px in LL (p<0.005); the GR in BB (p<0.02), BL (p<0.05) and LL (p<0.05); and the GSH in BL (p<0.01) and LL (p<0.001) was noted in comparison to the TT group. The GSH and GSH-Px were significantly low in LL (p<0.05) as compared to BT. A progressive decreasing trend in GSH level and enzyme activities was also noticed along the leprosy groups with advancing level of BI. The GSH, GSH-Px and GR were significantly low in BI levels 1.1-2 (p<0.005, p<0.05, p<0.02), 2.1-3 (p<0.005, p<0.001, p<0.005), 3.1-4 (p<0.005) and 4.1-6 (p<0.01, p<0.005, p<0.05) as compared to controls. A significant decline in GSH was noted in BI levels 1.1-2 (p<0.005), 2.1-3 (p<0.005), 3.1-4 (p<0.005) and 4.1-6 (p<0.01) as compared to the bacteriologically negative group. The GSH-Px (p<0.05) and GR (p<0.05) were significantly low in BI levels 2.1-3, 3.1-4 and 4.1-6 as compared to the bacteriologically negative group. CONCLUSION: The findings suggest oxidative stress associated with diminished antioxidant defence potential in leprosy. The study identifies association of diminished antioxidant potential with bacterial load and type of leprosy.

Erythrocyte superoxide dismutase, catalase activities and hydrogen peroxide induced lipid peroxidation in leprosy.

OBJECTIVES: To assess erythrocyte superoxide dismutase (SOD) and catalase (CAT) activities and hydrogen peroxide induced lipid peroxidation in leprosy. DESIGN: One hundred leprosy patients and 50 normal healthy controls were studied for the parameters. The data was analysed by grouping the patients into Ridley-Jopling (RJ) types [Tuberculoid leprosy (TT, n = 22), Borderline tuberculoid leprosy (BT, n = 28), Borderline leprosy (BB, n = 13), Borderline lepromatous leprosy (BL, n = 16) and Lepromatous leprosy (LL, n = 21)] and into different levels of Bacteriological Index (BI) [bacteriologically negative (n = 32), BI = 0.1-1 (n = 22), BI = 1.1-2 (n = 16), BI = 2.1-3 (n = 14), BI = 3.1-4 (n = 10) and BI = 4.1-6 (n = 06)]. RESULTS: The induced peroxidation was significantly high and the enzyme activities were significantly low in leprosy (total patients) as compared to controls. A progressive increase in peroxidation was detected along the leprosy spectrum from TT to LL and the increase was significant in BB, BL and LL groups as compared to controls. Induced peroxidation in LL group as compared to TT, BT and BB and in the BL group as compared to TT and BT were significantly different. A concomitant progressive decline in enzyme activity was detected along the leprosy spectrum from TT to LL. The SOD activity in BB, BL and LL and the CAT activity in BL and LL were significantly low as compared to controls. SOD activity in BB, BL and LL groups as compared to TT and in the LL group as compared to BT were significantly different. A progressive trend of increasing peroxidation and decreasing SOD and CAT activity were also detected along the leprosy groups with advancing level of BI. Induced peroxidation and SOD activity were significantly different in bacteriologically positive groups as compared to controls and in the BI levels 1.1-2, 2.1-3, 3.1-4 and 4.1-6 as compared to bacteriologically negative group. The peroxidation was significantly different in BI levels 2.1-3, 3.1-4 and 4.1-6 as compared to BI level 0.1-1. The CAT activity was significantly different in BI levels 2.1-3, 3.1-4 and 4.1-6 as compared to controls. CONCLUSION: The study findings suggest oxidative stressful state associated with reduced antioxidant defence potential in erythrocytes of leprosy patients. The study implicates association of erythrocyte oxidative stress with bacterial load and type of leprosy.

Undiagnosed purpura: a case of autoerythrocyte sensitization syndrome associated with dermatitis artefacta and pseudo-ainhum.

A 23-year-old young woman presented with recurrent episodes of painful bruising along with linear erosions on the accessible areas of the body of nine years duration with a pseudo-ainhum of her left nipple for the past three months. Her case history included repeated visits to various physicians at different centers and an extensive investigative profile. A diagnosis of autoerythrocyte sensitization was made on the basis of the clinical history, dermatological examination complemented by a positive autoerythrocyte sensitization test, psychiatric evaluation and absence of any organic cause for her ailment. She was placed on psychiatric management and has remained symptom-free after six months follow-up. The case is reported for its rarity, as well as for the association of autoerythrocyte sensitization syndrome with frank dermatitis artefacta and pseudo-ainhum, which to the best of our knowledge has not yet been reported in the literature.

Effect of listeriolysin O-loaded erythrocytes on Mycobacterium avium replication within macrophages.

OBJECTIVE: To evaluate the efficacy of erythrocytes loaded with the haemolytic toxin listeriolysin O against Mycobacterium avium replication within human macrophages. METHODS: Recombinant listeriolysin O was loaded in human erythrocytes by a procedure of hypotonic dialysis and isotonic resealing. Loaded erythrocytes were modified to allow them to be recognized and taken up by human macrophages infected with M. avium. The antimycobacterial activity of the erythrocytes loaded with listeriolysin O was evaluated by supernatant and intracellular cfu counts on days 4 and 7 post-erythrocyte administration. RESULTS: Recombinant listeriolysin O was encapsulated in human erythrocytes to reach final concentrations ranging from 1 to 4 ng/mL of erythrocytes. Erythrocytes loaded with increasing quantities of recombinant protein were able to reduce (at most by 50%) M. avium replication in a dose-dependent fashion when administered to infected macrophages. CONCLUSIONS: Erythrocytes loaded with listeriolysin O are effective against M. avium replication within macrophages. We are confident that the strategy presented could be useful against mycobacteria other than M. avium (such as Mycobacterium tuberculosis and Mycobacterium leprae) by itself or as part of an antimycobacterial treatment.

Hemolytic drugs aniline and dapsone induce iron release in erythrocytes and increase the free iron pool in spleen and liver.

Incubation of rat erythrocytes with the hydroxylated metabolites of aniline and dapsone (4-4'-diaminodiphenylsulfone), phenylhydroxylamine and dapsone hydroxylamine, respectively, induced marked release of iron and methemoglobin formation. On the contrary, no release of iron nor methemoglobin formation was seen when the erythrocytes were incubated with the parent compounds (aniline and dapsone). The acute intoxication of rats with aniline or dapsone induced a marked increase in the erythrocyte content of free iron and methemoglobin, indicating that the xenobiotics are effective only after biotransformation to toxic metabolites in vivo. Prolonged administration of aniline or dapsone to rats produced continuous release of iron from erythrocytes. Marked iron overload was seen in the spleen and in the liver Kupffer cells, as detected histochemically. The spleen weight in these subchronically treated animals was significantly increased. The free iron pool was markedly increased in the spleen and to a lower extent in the liver. The possible relationships between iron release in erythrocytes, oxidative damage seen in senescent cells, hemolysis, overwhelmed capacity of spleen and liver to keep iron in storage forms and subsequent increase in low molecular weight, catalitically active iron is discussed.

Copper-zinc superoxide dismutase from the marine yeast Debaryomyces hansenii.

We have isolated the cytosolic form of Cu-Zn superoxide dismutase (SOD) from the marine yeast Debaryomyces hansenii. This enzyme has a subunit mass of 18 kDa. The preparation was found to be heterogeneous by IF electrophoresis with two pI ranges: 5.14-4.0 and 1.6-1.8. The enzyme preparation had a remarkably strong stability at pH 6.0-7.0, surviving boiling for 10 min without losing more than 60% of activity. On Western blots, this enzyme was recognized by antibodies raised in rabbits against D. hansenii extracts, while only a weak cross-reaction could be detected using antibodies generated against either Saccharomyces cerevisiae or bovine erythrocyte Cu-Zn SODs. In sequencing analysis, a peptide obtained by trypsin digestion was found to have 85% identity to the S. cerevisiae Cu-Zn SOD.

Anti-inflammatory activity of Debaryomyces hansenii Cu,Zn-SOD.

BACKGROUND: Cu,Zn-superoxide-dismutase, Cu,Zn-SOD, can be obtained from different sources with different anti-inflammatory activities. In this study we compared the anti-inflammatory capacity of the marine yeast Debaryomyces hanseii Cu,Zn-SOD (Dh-SOD) with that of bovine erythrocytes (Be-SOD) in a preventive and a therapeutic fashion. METHODS: Edema was induced by carrageenan injection into the rat hind paw and was evaluated using a mercury plethysmograph. Development of the inflammatory process was followed by volume displacement at time 0 (carrageenan injection), 1, 2, 3, 4, 5, 6, 9, 12, and 24 h thereafter. Three different SOD doses were used in preliminary experiments to prevent edema: 10, 100, and 1,000 U/kg. RESULTS: The results indicate that, at the lowest dose (10 U/kg), both SOD samples are effective in reducing inflammation in both the prostaglandin and amplification phases (-24.8% and -17.5% in the case of Be-SOD, and 11.8% and -18.7% in the case of Dh-SOD, respectively) (p < 0.05). At 100 U/kg, Be-SOD also shows good anti-inflammatory activity in all edema phase (-27.1% in the serotonin phase; -19.4% in the prostaglandin phase; and -20% in the amplification phase) (p < 0.05), but Dh-SOD was less effective (-10.9%, -9.1%, and -5.7%). At the highest dose tested (1000 U/kg), Dh-SOD was, again, more effective than Be-SOD in all three edema phases (-33.1% and -1.5%; -17.9% and -2.6%; and -13.8% and 6.7%, respectively) (p < 0.05). When evaluated as a therapeutic alternative, single doses of Dh-SOD at 1,000 U/kg, and Be-SOD at 100 U/kg, both showed good anti-inflammatory activities (-31.7% and -23.5%, respectively) (p < 0.05). CONCLUSION: For therapy purposes alone, Dh-SOD appears to be a better anti-inflammatory agent than Be-SOD in carrageenan-induced edema.

Anti-Inflammatory activity od debaryomyces hansenii Cu, Zn-SOD

Background. Cu,Zn-superoxide-dismutase, Cu,Zn-SOD, can be obtained from different sources with different anti-inflammatory activities. In this study we compared the antiinflammatory capacity of the marine yeast Debaryomyces hanseii Cu,Zn-SOD (Dh-SOD) with that of bovine erythrocytes (Be-SOD) in preventive an a therapeutic fashion. Methods. Edema was induced by carrageenan injection into the rat hind paw and was evaluated using a mercury plethysmograph. Development of the inflammatory process was followed by volume displacement at time 0 (carrageenan injection), 1, 2, 3, 4, 5, 6, 9, 12, and 24 h thereafter. Three different SOD doses were used in preliminary experiments to prevent edema: 10, 100, and U/kg. Results. The results indicate that, at the lowest dose (10 U/kg), both SOD samples are effective in reducing inflammation in both the prostaglandin and amplification phases (-24.8 percent and -17.5 percent in the case of Be-SOD, and 11.8 percent and -18.7 percent in the case of Dh-SOD, respectively) (p<0.05). At 100 U/kg, Be-SOD also shows good anti-inflammatory activity in all edema phases (-27.1 percent in the serotonin phase; -19.4 percent in the prostaglandin phase; and -20 percent in the amplification phase) (p<0.05), but Dh-SOD was less effective (-10.9 percent, -9.1 percent, and -5.7 percent). At the highest dose tested (1000 U/kg), Dh-SOD was, again more effective than Be-SOD in all three edema phases (-33.1 percent and -1.5 percent; -17.9 percent and -2.6 percent; and -13.8 percent and 6.7 percent, respectively) (p >0.05). When evaluated as a therapeutic alternative, single doses of DH-SOD at 1,000 U/kg, and Be-SOD at 100 U/kg, both showed good anti-inflammatory activities (-31.7 percent and -23.5 percent, respectively) (p < 0.05). Conclusion. For therapy purposes alone, DH-SOD appears to be a better anti-inflammatory agent than Be-SOD in carrageenan-induced edema

Pesquisa de antígenos ABH em eritrócitos e na saliva de pacientes hansenianos / ABH antigens research in erythrocytes and at leprosy patients saliva

Os poucos estudos já publicados sobre a determinaçäo de fenótipo secretor dos antígenos ABH na saliva de hansenianos têm demonstrado näo haver uma correlaçäo significativa entre estas substâncias e a suscetibilidade à doença. No presente estudo avaliamos 74 pacientes, sendo 27 virchovianos, 23 tuberculóides e 24 dimorfos, quanto à presença de antíngenos ABH nos eritrócitos e na saliva, pela reaçäo de aglutinaçäo em tubo e inibiçäo de aglutinaçäo. A frequência dos grupos sanguineos ABO e do fenótipo secretor e näo secretor nestes pacientes e no grupo controle foram: O= 40,5(por cento) 49(por cento); AB= 6,8(por cento) 3,6(por cento) e näo secretor= 31,1(por cento) 17,5(por cento). Analisando-se os resultados apresentados nos hansenianos, observamos que näo houve antígenos ABH pesquisados nos eritrócitos e na saliva

Immunomodulatory assays to study structure-activity relationships of thalidomide.

Thalidomide, which has a long history of tragedy because of its ability to cause severe birth defects, is very effective in alleviating erythema nodosum leprosum in leprosy patients and aphthous ulcers in AIDS patients. The causes of these inflammatory diseases and the mechanism by which thalidomide diminishes them are unknown. It has been suggested that modulation of the immune response plays an important role. We found that thalidomide exerts immunomodulatory activity in three bioassays. It suppresses an IgM plaque forming cell response in mice injected with sheep erythrocytes: it inhibits TNF-alpha production by LPS stimulated human mononuclear cells: and it enhances IL-2 production by Con-A stimulated human mononuclear cells. We employed these bioassays to compare the activity of 15 analogs of thalidomide with thalidomide itself. Eight of the compounds were derivatives of the glutarimide moiety of thalidomide and the others were phthalimide or derivatives of the phthalimide moiety of thalidomide. N-hydroxyphthalimide, a simple derivative of phthalimide, was more effective than thalidomide and was also the most effective of the compounds assayed in suppressing the IgM plaque and TNF-alpha responses, but it did not enhance the IL-2 response, instead, it significantly suppressed it.

Combination of two mutant alpha spectrin alleles underlies a severe spherocytic hemolytic anemia.

We studied a patient with a severe spherocytic hemolytic anemia without family history of spherocytosis. Analysis of patient's erythrocyte membrane proteins revealed spectrin deficiency and a truncated alpha spectrin protein. We determined that the patient is a compound heterozygote with two mutations in alpha spectrin gene. Mutation in the paternal allele, designated alpha spectrin(PRAGUE), is a transition A to G in the penultimate position of intron 36 that leads to skipping of exon 37, frameshift, and production of the truncated alpha spectrin protein. The maternal allele, designated alpha spectrin(LEPRA), contains transition C-->T in position -99 of intron 30. This mutation enhances an alternative acceptor splice site 70 nucleotides upstream from the regular site. The alternative splicing causes a frameshift and premature termination of translation leading to a significant decrease in alpha spectrin production. The alpha(LEPRA) mutation is linked to a spectrin alphaIIa marker that was found to be associated with recessive or nondominant spectrin-deficient hereditary spherocytosis in approximately 50% of studied families. We conclude that the alpha(LEPRA) mutation combined in trans with the alpha(PRAGUE) mutation underlie the severe hemolytic anemia in the proband. We suggest that allele alpha spectrin(LEPRA) may be frequently involved in pathogenesis of recessive or nondominant spectrin-deficient hereditary spherocytosis.

Reversible oligonucleosome self-association: dependence on divalent cations and core histone tail domains.

Regularly spaced nucleosomal arrays equilibrate between unfolded and highly folded conformations in <2 mM MgCl2, and self-associate above 2 mM MgCl2 [Schwarz, P. M., & Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. Here we use analytical and differential sedimentation techniques to characterize the molecular mechanism and determinants of oligonucleosome self-association. Divalent cations induce self-association of intact nucleosomal arrays by binding to oligonucleosomal DNA and neutralizing its negative charge. Neither linker histones nor H2A/H2B dimers are required for Mg2+ - dependent self-association. However, divalent cations are unable to induce self-association of trypsinized nucleosomal arrays lacking their N- and C-terminal core histone tail domains. This suggests that the H3/H4 tail domains directly mediate oligonucleosome self-association through a non-Coulombic-based mechanism. Self-association occurs independently of whether the oligonucleosome monomers are folded or unfolded. The first step in the self-association pathway is strongly cooperative and produces a soluble association intermediate that sediments approximately 10 times faster than the oligonucleosome monomers. The size of the oligonucleosome polymers increases rapidly as a consequence of small increases in the divalent cation concentration, eventually producing polymeric species that sediment at >> 10 000 S. Importantly, all steps in the self-association pathway are freely reversible upon removal of the divalent cations. Taken together, these data indicate that short oligonucleosome fragments composed of only core histone octamers and DNA possess all of the structural features required to achieve chromosome-level DNA compaction. These findings provide a molecular basis for explaining many of the recently uncovered structural features of interphase and metaphase chromosomal fibers.

Aggregation and deformability of erythrocytes in leprosy.

The hemorheological parameters, erythrocyte aggregation and deformability are determined in leprotic patients and are compared with that of healthy subjects. The aggregation is determined by sequential analysis of the He-Ne laser transmission data through erythrocyte suspension at hematocrit 5%. The erythrocyte deformability is determined by measurement of passage time (reciprocal of deformability) of erythrocyte suspension in PBS at hematocrit 6% through cellulose membrane. The observations show that in leprosy the aggregation of erythrocyte is marginally reduced and the deformability is significantly increased. These parameters in combination with low hemoglobin and hematocrit levels in these patients lowers the blood viscosity to maintain the transport of material across the capillary wall.

Dapsone-induced hemolytic anemia.

Dapsone, an old drug introduced and used almost exclusively for the treatment of leprosy, is now utilized in an increasing number of therapeutic situations. However, its hemotoxicity is potentially severe and is often dose limiting. Effective countermeasures, based on resolution of the mechanisms underlying dapsone-induced hemotoxicity, could significantly enhance the therapeutic value of the drug. In studies on rat red cells, we have established that the N-hydroxy metabolites of dapsone, DDS-NOH and MADDS-NOH, are direct-acting hemolytic agents, that they are formed in amounts sufficient to account for the hemotoxicity of the parent drug, and that the action of these toxic metabolites in the red cell induces premature sequestration by the spleen. Incubation of rat red cells with hemolytic concentrations of arylhydroxylamines leads to the generation of hydroxyl, glutathiyl, and hemoglobinthiyl radicals, and the formation of protein-glutathione mixed disulfides. Disulfide-linked adducts are also formed between membrane skeletal proteins and hemoglobin monomers, as well as between the monomeric hemoglobin units forming dimers, trimers, tetramers, and pentamers. Profound morphological changes are seen with change from normal discoidocity to an extreme nonspherocytic enchinocyte shape. Parallel studies with human red cells indicate that the response of human cells is qualitatively similar but that there are notable differences in regard to skeletal membrane effects. A working hypothesis for the mechanism underlying dapsone hemolytic activity is proposed.