There is more breast-feeding in the professional classes than in the lower classes: and the Ministry report found that there were relatively more cot deaths among the babies of poor than of well-to-do parents, and among the babies of young and unmarried mothers than among those of the older and married. Moreover, there were fewer first children among the cot deaths than among the controls-another social factor perhaps related to the care and time which a mother can give to her child. Incidentally, the authors of this report found that a family history of allergy was commoner for the controls than for the cot deaths. Other workers57 have been unable to confirm the finding of Parish and colleagues on antibodies to cows' milk protein. R. M. Rothberg and R. S. Farr8 reported antibodies to cows' milk in the serum of 75% of children but could find no connexion between the presence of such antibodies and respiratory or other infections. E. Gold and F. C. Robins' have also reviewed the problem in the light of this work. In the B.M.7. this week Drs. J. M. Johnstone and H. S. Lawy describe a study of 56 cot deaths in the Grimsby area. They estimated the antibody titre to cows' milk protein in 37, and found a low titre in 5 cot deaths, the same proportion as in controls, and no antibodies in the remaining 32. On logical grounds it would seem to be extraordinarily unlikely that an anaphylactoid reaction would occur only when the baby is asleep. Probably all babies get some milk into the trachea. Why do they not die suddenly during feeds -surely the most likely time for sudden death to occur if those deaths are due to milk allergy ? Fully breast-fed babies suffer fewer respiratory infections than artificially fed ones.° 11 But the role of infection in causing cot deaths is uncertain. Drs. Johnstone and Lawy, though unable to isolate a virus in any of 47 cases, concluded from study of post-mortem cultures that a high proportion of cot deaths were due to pulmonary infection of the lower respiratory tract. They blamed in particular the pneumococcus, Klebsiella pneumoniae, and Staphylococcus pyogenes. M. A. Valdes-Dapena and colleagues12 13 attempted to isolate a virus from each of eight different sites in 109 cases of cot deaths, and detected a Coxsackie B5 virus in one case only. They found that the gamma-globulin content of the serum in 114 cases was normal. R. T. Cooke and R. G. Welch\"4 studied 122 cot deaths in the Hartlepools and found little evidence of overwhelming infection in most of them. They thought that most of the deaths were from anoxia due to laryngospasm, caused either by anaphylaxis or by neural spasm related to the autonomic nervous system. They were impressed by an unusually high incidence of cot deaths among twins, and concluded from a study of the surviving as well as the dead members of the twin pairs that infection played some part in causing death. The Ministry report' states that \"b acteriological and virological investigations were generally negative.\" Nevertheless, J. L. Emery' 15 found that careful questioning elicited a history of symptoms of disease at least 48 hours before death in some 95% of cases. He thought that most of these children died in the invasive phase of an infection. The fact remains that we do not know the cause of most cot deaths. The Ministry report' declared that a cause of death or possible cause can be found in one out of four cases. Valdes-Dapena\" 13 found a cause in 30 out of 109 cases, but some of these were questionable. Cooke and Welch\" found a reasonable cause in a quarter. Stowens' found a cause in 18%. As Emery3 points out, cot deaths are not confined to the home; they occur likewise in hospital, and it would be absolutely wrong to suggest to a bereaved, distraught mother that the death of her baby at home could have been prevented. Even if there were symptoms of a respiratory infection for 48 hours before the unexpected death, that does not mean that anything could have been done to prevent the death. Colds do not respond to antibiotics or any other medicine, and all children have colds. A cot death could have occurred in the family of any one of us, however careful we were. It is altogether wrong to make the mother feel guilty by telling her that the baby was suffocated, or would not have died if he had been breast-fed, or should have been taken to the doctor sooner-when we do not really know why he died.

Chemotherapy of tuberculosis.

Clofazimine is a poorly-soluble but orally-bioavailable small molecule drug that massively accumulates in macrophages when administered over prolonged periods of time. To determine whether crystal-like drug inclusions (CLDIs) that form in subcellular spaces correspond to pure clofazimine crystals, macrophages of clofazimine-fed mice were elicited with an intraperitoneal thioglycollate injection. Inside these cells, CLDIs appeared uniform in size and shape, but were sensitive to illumination. Once removed from cells, CLDIs were unstable. Unlike pure clofazimine crystals, isolated CLDIs placed in distilled water burst into small birefringent globules, which aggregated into larger clusters. Also unlike pure clofazimine crystals, CLDIs fragmented when heated, and disintegrated in alkaline media. In contrast to all other organelles, CLDIs were relatively resistant to sonication and trypsin digestion, which facilitated their biochemical isolation. The powder x-ray diffraction pattern obtained from isolated CLDIs was consistent with the diffraction pattern of liquid crystals and inconsistent with the expected molecular diffraction pattern of solid, three dimensional crystals. Observed with the transmission electron microscope (TEM), CLDIs were bounded by an atypical double-layered membrane, approximately 20 nanometers thick. CLDIs were polymorphic, but generally exhibited an internal multilayered organization, comprised of stacks of membranes 5 to 15 nanometers thick. Deep-etch, freeze-fracture electron microscopy of unfixed snap-frozen tissue samples confirmed this supramolecular organization. These results suggest that clofazimine accumulates in macrophages by forming a membrane-bound, multilayered, liquid crystal-like, semi-synthetic cytoplasmic structure.

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Macrophages Sequester Clofazimine in an Intracellular Liquid Crystal-Like Supramolecular Organization

(1995). The Pharmacology, Metabolism, and Chemistry of Clofazimine. Drug Metabolism Reviews: Vol. 27, No. 4, pp. 591-614.

The Pharmacology, Metabolism, and Chemistry of Clofazimine

Introduction. During the past two decades there has been a great increase in our knowledge of the mechanisms of virus multiplication in cultured cells. The cells utilized, however, have usually been undifferentiated standard cell lines, and little attention has been given to the interactions of viruses with differentiated cells, either in tissue culture or in the intact animal. Until recently, studies of viral pathogenicity have tended to be largely descriptive but a start has now been made in the analysis of the genetic and molecular control of virus pathogenicity for a number of model systems. Here we focus on one such system, Semliki Forest virus (SFV) infection of the laboratory mouse. This system has the initial advantages that the molecular biology of SFV and the closely related Sindbis virus has been extensively studied using standard tissue culture cell lines, that SFV is neurotropic, that strains of extremes of virulence for mice are available, and that inbred and immune-deficient mouse strains can be utilized.

Semliki Forest virus infection of mice: a model for genetic and molecular analysis of viral pathogenicity.

Clofazimine (CFZ) is an optically active, red-colored chemotherapeutic agent that is FDA approved for the treatment of leprosy and is on the World Health Organization's list of essential medications. Interestingly, CFZ massively accumulates in macrophages where it forms crystal-like drug inclusions (CLDIs) after oral administration of the drug in animals and humans. The analysis of the fluorescence spectra of CLDIs formed by resident tissue macrophages revealed that CFZ, when accumulated as CLDIs, undergoes a red shift in fluorescence excitation (from Ex: 540-570 to 560-600 nm) and emission (Em: 560-580 to 640-700 nm) signal relative to the soluble and free-base crystal forms of CFZ. Using epifluorescence microscopy, CLDI(+) cells could be identified, relative to CLDI(-) cells, based on a >3-fold increment in mean fluorescence signal at excitation 640 nm and emission at 670 nm. Similarly, CLDI(+) cells could be identified by flow cytometry, based on a >100-fold increment in mean fluorescence signal using excitation lasers at 640 nm and emission detectors >600 nm. CLDI's fluorescence excitation and emission was orthogonal to that of cell viability dyes such as propidium iodide and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), cellular staining dyes such as Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane), as well as many other fluorescently tagged antibodies used for immunophenotyping analyses. In vivo, >85% of CLDI(+) cells in the peritoneal exudate were F4/80(+) macrophages and >97% of CLDI(+) cells in the alveolar exudate were CD11c(+). Most importantly, the viability of cells was minimally affected by the presence of CLDIs. Accordingly, these results establish that CFZ fluorescence in CLDIs is suitable for quantitative flow cytometric phenotyping analysis and functional studies of xenobiotic sequestering macrophages.

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A far-red fluorescent probe for flow cytometry and image-based functional studies of xenobiotic sequestering macrophages.

In 1957 in a communication from these laboratories the remarkable antimycobacterial activity of B.663, 3-(P.-chloroanilino)-10-(P.-chlorophenyl)-2,10-dihydro-2-(isopropylimino)-phenazine, was first described (1). B.663, now known as Clofazimine, forms the active component of the antileprosy preparation Lamprene (Geigy).

A. G. Jina

Papers

The antileprosy agent clofazimine (b.663) in macrophages: light, electron microscope and function studies