Joyce T. Kawakami

Mitochondrial Swelling and Incipient Outer Membrane Rupture in Preapoptotic and Apoptotic Cells

Outer mitochondrial membrane (OMM) rupture was first noted in isolated mitochondria in which the inner mitochondrial membrane (IMM) had lost its selective permeability. This phenomenon referred to as mitochondrial permeability transition (MPT) refers to a permeabilized inner membrane that originates a large swelling in the mitochondrial matrix, which distends the outer membrane until it ruptures. Here, we have expanded previous electron microscopic observations that in apoptotic cells, OMM rupture is not caused by a membrane stretching promoted by a markedly swollen matrix. It is shown that the widths of the ruptured regions of the OMM vary from 6 to 250 nm. Independent of the perforation size, herniation of the mitochondrial matrix appeared to have resulted in pushing the IMM through the perforation. A large, long focal herniation of the mitochondrial matrix, covered with the IMM, was associated with a rupture of the OMM that was as small as 6 nm. Contextually, the collapse of the selective permeability of the IMM may precede or follow the release of the mitochondrial proteins of the intermembrane space into the cytoplasm. When the MPT is a late event, exit of the intermembrane space proteins to the cytoplasm is unimpeded and occurs through channels that transverse the outer membrane, because so far, the inner membrane is impermeable. No channel within the outer membrane can expose to the cytoplasm a permeable inner membrane, because it would serve as a conduit for local herniation of the mitochondrial matrix. Anat Rec, 2012.

A role for archaeal organisms in development of atherosclerotic vulnerable plaques and myxoid matrices

PURPOSE Vulnerable plaques are characterized by a myxoid matrix, necrotic lipidic core, reactive oxygen species, and high levels of microorganisms. Aerobic microbes such as Chlamydophila pneumoniae and Mycoplasma pneumoniae usually do not survive in oxidative stress media. Archaea are anaerobic microbes with powerful anti-oxidative enzymes that allow detoxification of free radicals whose presence might favor the survival of aerobic microorganisms. We searched for archaeal organisms in vulnerable plaques, and possible associations with myxoid matrix, chlamydia, and mycoplasma bodies. METHODS Twenty-nine tissue samples from 13 coronary artherectomies from large excentric ostial or bifurcational lesions were studied using optical and electron microscopy. Infectious agents compatible with archaea, chlamydia, and mycoplasma were semiquantified using electron micrographs and correlated with the amounts of fibromuscular tissue, myxoid matrix, and foam cells, as determined from semi-thin sections. Six of the cases were also submitted to polymerase chain reaction with archaeal primers. RESULTS All 13 specimens showed archaeal-compatible structures and chlamydial and mycoplasmal bodies in at least 1 sample. There was a positive correlation between extent of the of myxoid matrix and archaeal bodies (r = 0.44, P = 0.02); between archaeal and mycoplasmal bodies (r = 0.41, P = 0.03), and between chlamydial bodies and foam cells (r = 0.42; P = 0.03). The PCR test was positive for archaeal DNA in 4 of the 6 fragments. DISCUSSION DNA and forms suggestive of archaea are present in vulnerable plaques and may have a fundamental role in the proliferation of mycoplasma and chlamydia. This seems to be the first description of apparently pathogenic archaea in human internal organ lesions.

Do Archaea and bacteria co-infection have a role in the pathogenesis of chronic chagasic cardiopathy?

UNLABELLED Chronic cardiopathy (CC) in Chagas disease is a fibrotic myocarditis with C5b-9 complement deposition. Mycoplasma and Chlamydia may interfere with the complement response. Proteolytic enzymes and archaeal genes that have been described in Trypanosoma cruzi may increase its virulence. Here we tested the hypothesis that different ratios of Mycoplasma, Chlamydia and archaeal organisms, which are frequent symbionts, may be associated with chagasic clinical forms. MATERIALS AND METHODS eight indeterminate form (IF) and 20 CC chagasic endomyocardial biopsies were submitted to in situ hybridization, electron and immunoelectron microscopy and PCR techniques for detection of Mycoplasma pneumoniae (MP), Chlamydia pneumoniae(CP), C5b-9 and archaeal-like bodies. RESULTS MP and CP-DNA were always present at lower levels in CC than in IF (p < 0.001) and were correlated with each other only in CC. Electron microscopy revealed Mycoplasma, Chlamydia and two types of archaeal-like bodies. One had electron dense lipid content (EDL) and was mainly present in IF. The other had electron lucent content (ELC) and was mainly present in CC. In this group, ELC correlated negatively with the other microbes and EDL and positively with C5b-9. The CC group was positive for Archaea and T. cruzi DNA. In conclusion, different amounts of Mycoplasma, Chlamydia and archaeal organisms may be implicated in complement activation and may have a role in Chagas disease outcome.

Endocarditis Secondary to Microsporidia Giant Vegetation in a Pacemaker User

A 60-year-old white man was admitted with a 2-week history of shivering and fever (38°C). He had had a dual-chamber pacemaker implanted in 1996 because of complete atrioventricular block, with an elective pulse generator replaced 3 months before admission. Medications taken on a daily basis included amiodarone for atrial fibrillation rhythm control and atenolol for hypertension. His blood pressure was normal; his heart rate was 80 bpm; and he was febrile (38.5°C). Chest auscultation revealed a systolic murmur at the left and right sternal borders with respiratory shifting in the heart sound, not previously described in this patient. ECG showed a normally functioning pacemaker (Figure 1). Chest x-ray was normal (Figure 2), as were renal function and differential white cell count. Figure 1. Twelve-lead surface ECG showed a normally functioning dual-chamber pacemaker. Figure 2. A, Frontal anterior-posterior chest x-ray showed a pacemaker in the right infraclavicular region with no other abnormality. B, Normal frontal posterior-anterior chest x-ray after surgical intervention. Endocarditis was confirmed by a transesophageal echocardiography …A 60-year-old white man was admitted with a 2-week history of shivering and fever (38°C). He had had a dual-chamber pacemaker implanted in 1996 because of complete atrioventricular block, with an elective pulse generator replaced 3 months before admission. Medications taken on a daily basis included amiodarone for atrial fibrillation rhythm control and atenolol for hypertension. His blood pressure was normal; his heart rate was 80 bpm; and he was febrile (38.5°C). Chest auscultation revealed a systolic murmur at the left and right sternal borders with respiratory shifting in the heart sound, not previously described in this patient. ECG showed a normally functioning pacemaker (Figure 1). Chest x-ray was normal (Figure 2), as were renal function and differential white cell count. Figure 1. Twelve-lead surface ECG showed a normally functioning dual-chamber pacemaker. Figure 2. A, Frontal anterior-posterior chest x-ray showed a pacemaker in the right infraclavicular region with no other abnormality. B, Normal frontal posterior-anterior chest x-ray after surgical intervention. Endocarditis was confirmed by a transesophageal echocardiography …

Infection and Microparticles may Cause Complication of Atherosclerotic Plaques

Atherosclerosis is frequently associated with diabetes, obesity, metabolic syndrome and oxidized low density lipoproteins (oxLDL). Many studies have reported association of infection with such disorders, but with controversial results. In our view, these findings have relationship with technical differences. We showed previously, presence of infectious agents inside of ruptured plaque, using immunohistochemistry and electron microscopy. More recently, we detected Electron Lucent microparticles (ELMP) and Mycoplasma pneumoniae (Mp) lipoproteins in vulnerable plaques (VP). However, we have interest to know if ELMPs contain Mp lipoprotein antigens and if they are related to oxLDL and plaque vulnerability. Methods: We studied three groups of coronary arteries: VP (vulnerable plaque; n=13), stable plaques (SP; n=7), and normal arteries (NA; n=7). All cases were studied by immuno electron microscopy, and the mean numbers of ELMPs, oxLDL and Mp antigens, inside and outside ELMP, were obtained. Double colloidal immunogold particles (anti-oxLDL and anti-Mp) allowed the simultaneous localization of both antigens. Results: There was a significant higher amount of ELMPs in VP, with positive dots for both oxLDL and Mp antigens inside them, compared to other two groups (p<0.01). Mp and oxLDL antigens were co-localized in lipidic nanoparticles intra ELMPs, showing positive correlation (r=0.60; P=0.04). High amount of oxLDL and Mp antigens extra ELMPs were seen in VP, but not in stable plaques. Conclusion: Plaque vulnerability in atherosclerosis may be related to presence of ELMPs, containing M. pneumoniae lipoproteins and oxLDL. We hypothesized that M. pneumoniae lipoproteins oxidation could be a mechanism for this association. However, further data are necessary to prove this hypothesis.

Comparison of the Protective Effects of Individual Components of Particulated trans-Sialidase (PTCTS), PTC and TS, against High Cholesterol Diet-Induced Atherosclerosis in Rabbits

Previous studies showed the presence of Mycoplasma pneumoniae (M. pneumoniae) and membrane-shed microparticles (MPs) in vulnerable atherosclerotic plaques. H&S Science and Biotechnology developed PTCTS, composed by natural particles from medicinal plants (PTC) combined with trans-Sialidase (TS), to combat MPs and Mycoplasma pneumoniae. Our aim was to determine the effects of the different components of PTCTS in a rabbit model of atherosclerosis. Rabbits were fed with high cholesterol diet for 12 weeks and treated during the last 6 weeks with either vehicle, PTC, TS, or PTCTS. Lipid profile and quantification of MPs positive for Mycoplasma pneumoniae and oxidized LDL antigens were carried out. Aortas and organs were then histologically analyzed. PTCTS reduced circulating MPs positive for Mycoplasma pneumoniae and oxidized LDL antigens, reduced the plaque area in the abdominal aorta, and caused positive remodeling of the ascendant aorta. PTC caused positive remodeling and reduced plaque area in the abdominal aorta; however, TS had a lipid lowering effect. PTCTS components combined were more effective against atherosclerosis than individual components. Our data reinforce the infectious theory of atherosclerosis and underscore the potential role of circulating MPs. Therefore, the removal of Mycoplasma-derived MPs could be a new therapeutic approach in the treatment of atherosclerosis.

Loose and compact agglomerates of 50 nm microvesicles derived from Golgi and endoplasmic reticulum membranes in pre- and in -apoptotic Mycoplasma infected HeLa cells: host-parasite interactions under the transmission electron microscope.

Sao Paulo, November 17, 2014Dear Editor The fine structure of apoptotic HeLa cells from cultures contaminated with mycoplasma in early and in advanced stages of the cell demise process differs from those so far described in apoptotic cells. The observed changes are enhanced after exposure of the cells to staurosporine. At low microscopic magnifications cells that have apparent normal cytoplasm and nuclei, actually may be harbouring cystic-like profile(s) of parasitic origin in an altered cytoplasm. The membranes of the transitional elements of the endoplasmic reticulum (TER) appear fragmented in irregular branching stripes of the smooth component of the TER (Fig. 1, white asterisks in L delimited area). The concentration of the rough endoplasmic reticulum (RER) membranes is less than in normal HeLa cells. Near to the smooth ER tubule-saccular elements lie groups of 50 nm microvesicles aside stacked, thin, various sized profiles of Golgi saccules (

Images in cardiovascular medicine. Endocarditis secondary to microsporidia: giant vegetation in a pacemaker user.

A 60-year-old white man was admitted with a 2-week history of shivering and fever (38°C). He had had a dual-chamber pacemaker implanted in 1996 because of complete atrioventricular block, with an elective pulse generator replaced 3 months before admission. Medications taken on a daily basis included amiodarone for atrial fibrillation rhythm control and atenolol for hypertension. His blood pressure was normal; his heart rate was 80 bpm; and he was febrile (38.5°C). Chest auscultation revealed a systolic murmur at the left and right sternal borders with respiratory shifting in the heart sound, not previously described in this patient. ECG showed a normally functioning pacemaker (Figure 1). Chest x-ray was normal (Figure 2), as were renal function and differential white cell count. Figure 1. Twelve-lead surface ECG showed a normally functioning dual-chamber pacemaker. Figure 2. A, Frontal anterior-posterior chest x-ray showed a pacemaker in the right infraclavicular region with no other abnormality. B, Normal frontal posterior-anterior chest x-ray after surgical intervention. Endocarditis was confirmed by a transesophageal echocardiography …A 60-year-old white man was admitted with a 2-week history of shivering and fever (38°C). He had had a dual-chamber pacemaker implanted in 1996 because of complete atrioventricular block, with an elective pulse generator replaced 3 months before admission. Medications taken on a daily basis included amiodarone for atrial fibrillation rhythm control and atenolol for hypertension. His blood pressure was normal; his heart rate was 80 bpm; and he was febrile (38.5°C). Chest auscultation revealed a systolic murmur at the left and right sternal borders with respiratory shifting in the heart sound, not previously described in this patient. ECG showed a normally functioning pacemaker (Figure 1). Chest x-ray was normal (Figure 2), as were renal function and differential white cell count. Figure 1. Twelve-lead surface ECG showed a normally functioning dual-chamber pacemaker. Figure 2. A, Frontal anterior-posterior chest x-ray showed a pacemaker in the right infraclavicular region with no other abnormality. B, Normal frontal posterior-anterior chest x-ray after surgical intervention. Endocarditis was confirmed by a transesophageal echocardiography …

Response to Letter Regarding Article, “Endocarditis Secondary to Microsporidia: Giant Vegetation in a Pacemaker User”

We appreciate Dr Henrikson’s interest in our study1 and the opportunity to clarify several issues. Dr Henrikson had a very precise observation concerning the electrocardiogram, and we agree with the comments made. Indeed, it was a normofunctioning dual-chamber pacemaker, and the electrocardiogram was taken just before open heart surgery, with the pacemaker programmed to asynchronous ventricular pacing mode considering atrial lead manipulation. Moreover, in our article, we report a novel agent for implantable pacemaker infection in a patient with persistent bacteremia despite the fact that blood cultures were all negative, as highlighted by Dr Henrikson. Most cases of pacemaker lead infections are due to pathogens from the skin flora, with positive blood culture ranging in the literature from 80% to 100% of the cases. Making a parallel with heart valves in the human, there are many causes of culture-negative endocarditis. The causative agents of culture-negative endocarditis in the heart are fastidious bacteria (Bartonella quintana, Coxiella burnetii, or brucella species), fungi, and the usual organisms (mainly streptococci) found in patients who have received antibiotic treatment before blood samples are obtained for culture.2 Thus, we present here another possible etiology in this context, now related to pacemaker infections. While these fastidious bacteria yield negative hemocultures requiring different diagnostic approaches such as serology, in the case of microsporidium, electron microscopy is fundamental and should be complemented by polymerase chain reaction.1 Dr Henrikson also pointed out that no signs of endocarditis were present even though a large vegetation was disclosed. Pacemaker endocarditis may be limited to the pacemaker lead or involve the cardiac valves, generally the tricuspid, which can be demonstrated by the echocardiogram or intraoperatively. In our case there was no vegetation in this valve by both methods, but the mass entailed in valve insufficiency, present in 25% of the cases in one series.3 We cannot rule out that microscopic endocarditis was present because no biopsy specimens were taken from the valve. Nonetheless, the good clinical outcome after a long-term follow-up period makes this possibility unlikely.

Endocarditis Secondary to Microsporidia

A 60-year-old white man was admitted with a 2-week history of shivering and fever (38°C). He had had a dual-chamber pacemaker implanted in 1996 because of complete atrioventricular block, with an elective pulse generator replaced 3 months before admission. Medications taken on a daily basis included amiodarone for atrial fibrillation rhythm control and atenolol for hypertension. His blood pressure was normal; his heart rate was 80 bpm; and he was febrile (38.5°C). Chest auscultation revealed a systolic murmur at the left and right sternal borders with respiratory shifting in the heart sound, not previously described in this patient. ECG showed a normally functioning pacemaker (Figure 1). Chest x-ray was normal (Figure 2), as were renal function and differential white cell count. Figure 1. Twelve-lead surface ECG showed a normally functioning dual-chamber pacemaker. Figure 2. A, Frontal anterior-posterior chest x-ray showed a pacemaker in the right infraclavicular region with no other abnormality. B, Normal frontal posterior-anterior chest x-ray after surgical intervention. Endocarditis was confirmed by a transesophageal echocardiography …A 60-year-old white man was admitted with a 2-week history of shivering and fever (38°C). He had had a dual-chamber pacemaker implanted in 1996 because of complete atrioventricular block, with an elective pulse generator replaced 3 months before admission. Medications taken on a daily basis included amiodarone for atrial fibrillation rhythm control and atenolol for hypertension. His blood pressure was normal; his heart rate was 80 bpm; and he was febrile (38.5°C). Chest auscultation revealed a systolic murmur at the left and right sternal borders with respiratory shifting in the heart sound, not previously described in this patient. ECG showed a normally functioning pacemaker (Figure 1). Chest x-ray was normal (Figure 2), as were renal function and differential white cell count. Figure 1. Twelve-lead surface ECG showed a normally functioning dual-chamber pacemaker. Figure 2. A, Frontal anterior-posterior chest x-ray showed a pacemaker in the right infraclavicular region with no other abnormality. B, Normal frontal posterior-anterior chest x-ray after surgical intervention. Endocarditis was confirmed by a transesophageal echocardiography …