Recognition of important roles of gangliosides in normal and abnormal cell function has motivated pharmacological modification of cellular ganglioside content. However, constitutive depletion of gangliosides in untransformed human cells has not been reported. In this context, the recent identification of a kindred carrying a point mutation in the GM3 synthase [ST3Gal5, Siat9] gene (Simpson et al, Nature Genetics 36:1225-9, 2004) provided an opportunity to explore this possibility. We established primary cultures of skin fibroblasts of three patients homozygous for this autosomal recessive defect. They exhibited a 93% reduction in ganglioside content (0.8+/-0.2 nmol LBSA per 10(7) cells versus 12.7+/-1.3 nmol LBSA per 10(7) normal fibroblasts). Importantly, this marked reduction was not compensated by activation of an alternate pathway of ganglioside synthesis, as occurs in murine GM3 synthase knockout fibroblasts. Cell morphology appeared unaffected, but under stringent conditions EGF-induced proliferation and migration of the mutant fibroblasts were reduced by 80% and 60%, respectively. Probing potential explanations, we found that EGF binding (effective membrane EGF receptor (EGFR) number) was reduced by 52%(to 6.2+/-1.9 from 12.8+/-2.0 pmol/10(8) normal fibroblasts, p<0.01), despite normal total EGFR protein. EGFR activation was likewise reduced as was EGF-induced Rho/Rac1 phosphorylation, which is associated with cell migration. We conclude that this GM3 synthase point mutation almost completely depletes human fibroblast cellular gangliosides, dampens membrane EGFR activation, and modulates related critical cell functions such as proliferation and migration. These cells offer a valuable model for the study of ganglioside modulation of cell function.

Psychotic symptoms such as delusions and hallucinations can have a devastating effect on a patient's social functioning. Since psychosis is rarely congenital, it is possible that lifestyle factors play a role in its etiology. This paper offers a hypothesis that some of these factors could be:(a) A lifestyle lacking evolutionarily conserved stressors such as frequent exposure to heat and/or cold, resulting in a lack of "thermal exercise" which could lead to malfunctioning of the brain.(b) Partial retention and absorption of toxic waste in the colon, as described in more detail below.(c) Genetic makeup that makes a person vulnerable to the above conditions. To test the hypothesis, three types of hydrotherapy are proposed (to be tested separately) as a putative neuroleptic treatment: head-out hot showers, adapted cold showers (twice daily each), and colon hydrotherapy (every 3-12 weeks, which also includes a dietary change according to Harvard's Healthy Eating Pyramid). The following is supporting evidence: Dopaminergic transmission in the mesolimbic pathway is involved in central processing of pain and negative stimuli (e.g. stress-induced analgesia) in addition to its role in the pathophysiology of psychosis. It is also known that if a neural pathway can perform two different functions, then the execution of one function will often suppress the other (e.g. gate control theory of pain). Thus, a pain-based therapy, such as a moderately hot shower, could have a "crowding out" effect on pathological processes within the mesolimbic system. In addition, hyperthermia is known to induce fatigue and depress activity of the frontal cortex (the sedative effect). As described previously, an adapted cold shower could work as a mild electroshock applied to the sensory cortex and, therefore, it might have an antipsychotic effect similar to that of electroconvulsive therapy. Additionally, a cold shower is a vivid example of stress-induced analgesia and would also be expected to "crowd out" or suppress psychosis-related neurotransmission within the mesolimbic system. Human and bacterial toxic waste can sometimes be partially retained in the colon and it is known that many high-molecular-weight compounds can be absorbed there. Most narcotics can cause intoxication if administered rectally and there is also significant comorbidity of schizophrenia with intestinal illnesses. Additionally, there is indirect evidence that colon cleansing can significantly improve mental state. Therefore, it is possible that chronic intoxication with yet unknown components of partially retained waste could be one of the unrecognized organic causes of psychosis.

To probe the functions of membrane gangliosides, the availability of ganglioside-depleted cells would be a valuable resource. To attempt to identify a useful genetic model of ganglioside depletion, we assessed ganglioside metabolism in murine GM3 synthase (GM3S)-/- knockout primary embryonic fibroblasts (MEF), because normal fibroblast gangliosides (GM3, GM2, GM1, and GD1a), all downstream products of GM3S, should be absent. We found that heterozygote MEF (GM3S+/-) did have a 36% reduced content of qualitatively normal gangliosides (7.0+/-0.8 nmol LBSA/mg cell protein; control: 11+/-1.6 nmol). However, two unexpected findings characterized the homozygous (GM3-/-) MEF. Despite complete knockout of GM3S,(i) GM3-/- MEF retained substantial ganglioside content (21% of normal or 2.3+/-1.1 nmol) and (ii) these gangliosides were entirely different from those of wild type MEF by HPTLC. Mass spectrometry identified them as GM1b, GalNAc-GM1b, and GD1alpha, containing both N-acetyl and N-glycolylneuraminic acid and diverse ceramide structures. All are products of the 0 pathway of ganglioside synthesis, not normally expressed in fibroblasts. The results suggest that complete, but not partial, inhibition of GM3 synthesis results in robust activation of an alternate pathway that may compensate for the complete absence of the products of GM3S.

BACKGROUND: The erythrocyte sedimentation rate (ESR) is a simple and inexpensive laboratory test, which is widespread in clinical practice, for assessing the inflammatory or acute response. This work addresses the theoretical and experimental investigation of sedimentation a single and multiple particles in homogeneous and heterogeneous (multiphase) medium, as it relates to their internal structure (aggregation of solid or deformed particles). METHODS: The equation system has been solved numerically. To choose finite analogs of derivatives we used the schemes of directional differences. RESULTS:(1) Our model takes into account the influence of the vessel wall on group aggregation of particles in tubes as well as the effects of rotation of particles, the constraint coefficient, and viscosity of a mixture as a function of the volume fraction.(2) This model can describe ESR as a function of the velocity of adhesion of erythrocytes;(3) Determination of the ESR is best conducted at certain time intervals, i.e. in a series of periods not exceeding 5 minutes each;(4) Differential diagnosis of various diseases by means of ESR should be performed using the aforementioned timed measurement of ESR;(5) An increase in blood viscosity during trauma results from an increase in rouleaux formation and the time-course method of ESR will be useful in patients with trauma, in particular, with traumatic shock and crush syndrome. CONCLUSION: The mathematical model created in this study used the most fundamental differential equations that have ever been derived to estimate ESR. It may further our understanding of its complex mechanism.

BACKGROUND: Blunt trauma causes short-term compression of some or all parts of the chest, abdomen or pelvis and changes hemodynamics of the blood. Short-term compression caused by trauma also results in a short-term decrease in the diameter of blood vessels. It has been shown that with a sudden change in the diameter of a tube or in the direction of the flow, the slower-moving fluid near the wall stops or reverses direction, which is known as boundary layer separation (BLS). We hypothesized that a sudden change in the diameter of elastic vessel that results from compression may lead not only to BLS but also to other hemodynamic changes that can damage endothelium. METHODS: We applied Navier-Stokes, multiphase and boundary layer equations to examine such stress. The method of approximation to solve the BL equations was used. Experiments were conducted in an aerodynamic tube, where incident flow velocity and weight of carriage with particles before and after blowing were measured. RESULTS: We found that sudden compression resulting from trauma leads to (1) BLS on the curved surface of the vessel wall;(2) transfer of laminar boundary layer into turbulent boundary layer. Damage to the endothelium can occur if compression is at least 25% and velocity is greater than 2.4 m/s or if compression is at least 10% and velocity is greater than 2.9 m/s. CONCLUSION: Our research may point up new ways of reducing the damage from blunt trauma to large vessels. It has the potential for improvement of safety features of motor vehicles. This work will better our understanding of the precise mechanics and critical variables involved in diagnosis and prevention of blunt trauma to large vessels.

A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research.

As this paper goes to press a complete review of the chemistry of the fertile egg will be appearing (19). The author, Mr. J. Needham, was kind enough to allow me to inspect his manuscript and thus avail myself of the comprehensive bibliography and discussion. It is surprising that no biochemists have estimated the changing water content of the egg during incubation. Many of the analyses reported in Needham's review were expressed in per cent of total weight or per cent of dry solid, and consequently are of questionable value, since these latter functions are themselves changing; the former due to water evaporation and the latter through the addition of shell constituents and the burning of oxidizable organic compounds. Moreover, there has been no statistical treatment of the results, and the reliability of the average, figures obtained has consequently been difficult to estimate. Tangl's work, quoted throughout this paper, except for its lack of statistical treatment is more enlightening. However, his concept of the so called "Energy of Embryogenesis" which he propounds, seems to me misleading and unwarranted. What Tangl measured was the amount and the caloric value of the solid material burned and thus the quantity of energy lost during the embryonic period. The latter is equivalent to the usual measurements of catabolism. In the case of the embryo it is not basal metabolism which is being estimated, since the conditions are not basal. The embryo is absorbing and assimilating nutriment all the while at a relatively rapid rate. The calorific value of the oxidized solid, which is in truth the amount of energy lost during a certain chosen interval, in Tangl's judgment stands for the energy of embryogenesis; i.e., the energy of development (growth + differentiation). We believe that this conception is erroneous. The two processes, anabolism and catabolism, occur together and undoubtedly have some relationship, but surely one is not a measure of the other. In a starving animal, and so probably in a starving embryo, there is a considerable amount of so called basal metabolism. Thus if the "Embryogenetic Energy" were measured under these conditions a figure would be obtained for which there was no growth to correspond, or in other words there would be a value for something which did not exist. It will be seen in our later communications that the changes with age of metabolic rate and growth rate do not coincide. The amount of catabolism under certain circumstances does not accelerate growth or anabolism, but seems rather to be a limiting factor. It is as if when the absorbed energy were constant an increase of catabolism would make inroads upon the amount of energy which otherwise would remain for storage (growth). If, as Pembrey's (20) experiments would tend to show, there is an increase of metabolism in the oldest embryos when the outside temperature is lowered, one would find at the end of incubation in such cases that there was a greater amount of so called "Energy of Development" but smaller embryo. It seems that the potential energy amassed as growth comes from that remaining after the needs of the body have been satisfied. The results of the experiments described in this paper have formed the basis for judgment in the selection of suitable standard conditions for the incubation of hen's eggs. Standardization was necessary so that in future experiments the more important environmental factors might be kept uniform within a certain appropriate range and therefore not be held accountable for deviations observed in the embryos. Henceforth in this series of papers the term "standard incubation conditions" will signify that (1) the temperature was constantly at 38.8 +/- 0.4 degrees C.,(2) the humidity at 67.5 +/- 2.5 per cent,(3) there was a continuous flow of warm air into the incubator to provide the necessary circulation, and (4) the eggs were rolled once a day within the constant temperature room. The incubator, a double-walled copper cabinet, stands in a constant temperature room, the fluctuations of which are +/- 1.0 degrees C. The space between the walls of the incubator is filled with water which serves as a buffer to outer variations. It might be repeated that all the eggs are from White Leghorn hens, are incubated 2 days after laying, and that they are kept cold during the interval necessary for transportation. With the figures from our chemical analyses and metabolic rate experiments, it was possible to calculate values for the concentration of total solids, fat, and nitrogen throughout the incubation period. These data were necessary as a general chemical background for further work. The results of the calculations are obviously rough. Because of the great variability of the eggs a satisfactory degree of accuracy could not have been attained without a very large number of analyses supplemented by complete statistical treatment. The necessity for such a comprehensive study was not evident, and it is our belief that the approximations reached in this paper are sufficiently close to serve our present purposes. The chief facts that have been ascertained in this investigation are (1) Loss of water by the egg during incubation is a function of the atmospheric humidity in its immediate environment. More rapid circulation of air lowers the humidity around the egg and thus increases evaporation. Other facts influencing evaporation are (a) atmospheric temperature,(b) thickness and surface area of the shell, and (c) conditions within the egg, the most important of which, it is suggested, is the amount of heat produced by the embryo. The latter factor, in turn, depends upon its size and age, and a significant change does not become apparent until the last 3 or 4 days of incubation, that is to say, when the embryo is of sufficient mass to exert a measurable force.(2) The surface area of the eggs in sq. cm. may be approximately represented by the formula S = K W(2/3), where K = 5.07 +/- 0.10, and W = the weight of the whole egg in gm.(3) There is a loss of weight by the shell during incubation. This is most noticeable near the end of the cycle, when the loss seems to parallel in general the weight of the embryo.(4) There is also a loss of solid matter during incubation. Chemical analyses indicate that about 98 per cent of the material oxidized is fat. This conclusion is corroborative of previous work by Hasselbalch, Hasselbalch and Bohr, and Tangl.(5) Carbon dioxide may be measured with relative accuracy. When it is assumed that it is derived from the oxidation of fat, satisfactory corroboration of the chemical analyses is obtained. These experiments have furnished the data from which the values have been calculated for total solids, fats, and protein in the whole egg throughout incubation. The figures may be used later for comparison with the concentration of these substances within the embryo.

It will be well to restate the main problem at this point and to examine how far the accumulated data can help to elucidate it. The problem is this: Why are old mice generally resistant to all forms of peripheral inoculation of vesicular stomatitis virus when intracerebral injection is equally fatal for mice of all ages? The results of experiments in which the presence of virus was demonstrated by animal passage suggested that the reason can perhaps be found in (a) the different mechanisms of virus progression after intracerebral and peripheral injection, and (b) the development with age of localized barriers capable of halting the spread of virus (1, 2). The present study sought histological evidence for the nature of virus progression and for the changes observed in the older animals. The results clearly demonstrate that after intracerebral injection virus spreads along an open system, the lesions being distributed almost entirely in contiguity with the ventricles and their extensions, while after peripheral inoculations the evidence points to progression of the virus in a closed system of neurons and their processes, at least in the stage preceding neuronal necrosis, the distribution of lesions depending upon the central connections of the primary neurons connected with the inoculated site. Thus, in young mice, nasal instillation of the virus was followed by necrosis of a long chain of neurons, starting with those in the olfactory mucosa and progressing through specific zones of the olfactory pathway, pursuing the same order in which the various regions are known to have their major connections with one another. It is important to note that after nasal instillation the apparent lesions were present where the cell bodies of the neurons are situated, and not along the tracts connecting one group of neurons with another, which accounts for the lack of contiguity between the affected zones and the normal appearing, intervening areas. The assumption that the primary progression of the virus in this case occurs in a closed system is based on the absence of lesions in unrelated areas contiguous to those which are necrotic and to the tracts which connect one affected zone with another. Additional evidence for the assumption that the initial dissemination of peripherally injected virus is in a closed system is found in the decussating optic nerve pathway primarily pursued by the intraocularly injected virus. The progression of the virus along this decussating pathway was indicated in the experimental data obtained on mice 21 days or older, while in younger animals the spread of virus was so rapid and diffuse that the pathways along which it might have occurred remained obscure (2). In the present study, in which 15 day old mice were used, the lesions in the retinal neurons and the constant involvement of only the contralateral superior colliculus left little doubt that the primary spread of the virus, even in these very young animals, must have occurred within the retinal neuron processes (axons) which decussate in the optic chiasm (in the mouse, as in the rat, very few of these go to the homolateral side) and synapse chiefly with the neurons of the contralateral superior colliculus and also, apparently to a lesser extent, with those of the contralateral external geniculate body, where lesions were also demonstrated. Virus spreading in the optic nerve along the perineural subarachnoid space would be found at the base of the brain at the optic chiasm; virus extending along the interstitial spaces in the optic nerve should involve not only the nuclei of both sides of the optic pathway but also non-optic structures, such as the medial geniculate bodies, posterior colliculi, etc., by means of the commissures of von Gudden and of Meynert, whose fibers course through the chiasm. The highly specific localization observed in the present study is best accounted for by progression along the suggested closed pathway. Hurst (10) observed that poliomyelitis virus, after injection into the left sciatic nerve, may, after invading the lumbar cord, be found first in the contralateral motor cortex or thalamus and he suggested that this was evidence of progression along a decussating pathway and in favor of the axonal hypothesis of virus spread. It was not shown, however, that this particular localization was specifically related to the introduction of virus in the left sciatic nerve, or that it could be reversed by inoculating the sciatic nerve of the opposite side. The hypothesis proposed by Hurst, however, finds support in the present instance for (a) the superior colliculi never showed lesions after intracerebral, intranasal, or intramuscular inoculations, and (b) necrosis was produced in either the right or the left superior colliculus, depending on whether the virus was injected into the left or right eyes. The localization of lesions after injection of virus into the muscles of one leg indicated that in the young the invasion occurred along the local peripheral nerves, especially the motor fibers (neurons destroyed in the lumbar cord with those in the spinal ganglia intact), after a primary attack on the muscle itself. The only other lesions found at a late stage were in the reticular substance of the medulla, the olfactory portions of the brain appearing entirely normal. In this respect the mechanism of progression of intramuscularly injected vesicular stomatitis virus differs from that of eastern equine encephalomyelitis and pseudorabies viruses similarly injected into mice of the same age and breed: the former (E.E.E.) invades the central nervous system in the majority of instances, by being eliminated on the nasal mucosa and then along the olfactory pathways (18), while the latter appears to employ chiefly the local sensory fibers, attacking primarily the neurons in the spinal ganglia (unpublished observations). Because the CNS of old mice remain for the most part susceptible to vesicular stomatitis virus (although definite evidence of resistance to necrosis of the neurons was observed), and because after intracerebral injection the virus has been shown to spread in an open (ventricular) system, it is clear why young and old mice are equally susceptible to inoculation by this route. After peripheral inoculation, however, it has been amply demonstrated by experimental and histological methods that the spread of this virus begins and continues, at least until the cells disintegrate, in a closed system within the neurons and their processes and apparently also across the synapses. The halting of the virus somewhere in the anterior rhinencephalon after nasal instillation in resistant mice (1) would appear to be due to an arrest in an insusceptible neuron or an impenetrable synapse somewhere in the chain, and to the failure of the affected neurons to disintegrate (no lesions were found in the CNS of these mice) and thus to liberate the virus into the open system. After intramuscular injection, on the other hand, the virus encounters a different kind of muscle cell in the old mouse, and its inability to invade the nerves may perhaps be bound up with its demonstrated inability to attack and multiply in these changed muscle cells, although the role of a possible alteration in the terminal nerve endings themselves is not yet clear. After intraocular injection, the virus fails to affect visibly the retinal neurons of resistant old mice and the further invasion of the CNS is inhibited. The resistance of old mice to peripheral inoculations of vesicular stomatitis virus thus appears to be the result of (a) changes produced by age not in the whole animal but in certain specific, isolated structures, and (b) the special mode of progression of peripherally injected virus. It may be of interest to point out two phenomena which may perhaps be related to the one investigated in the present study. Tobacco mosaic virus has been found to produce different types of disease in certain plants of different ages; thus a widespread, systemic necrosis leads to the death of young Nicotiana rustica plants, while in old plants it is possible to produce necrotic foci in many parts of the plant by direct inoculation, although generalization does not occur from an isolated focus as it does in young specimens (19). In other words, age apparently does not change the whole plant, but it does transform something which allows the virus to spread easily from one site to another. MacNider (20) has observed that dogs which survive a severe type of hepatic injury from uranium, repair this injury with a special type of atypical, epithelial cell and become resistant not only to secondary intoxications by uranium but also by chloroform; he has also found that this change in epithelial cell type may be acquired as a product of senility, and that when it develops it imparts to the liver a degree of resistance to chloroform comparable to that induced by a process of repair following a severe hepatic injury from uranium nitrate.

In the present condition of the technique of cultivation of tissues, the only possible way of studying leucocytic secretions was to grow colonies of leucocytes in a medium of known properties and to examine the modifications of these properties under the influence of the living cells. The method was far from perfect, because the secretions were mixed with serum and accumulated for 48 hours in a medium where they probably underwent partial destruction. But an approximate idea of certain of the qualities of the secretions, although not of their quantity, could be derived from the experiments. In the fluids extracted from the cultures, we attempted to detect the presence of the leucocytic secretions through their physiological effects on homologous and foreign cells. Two kinds of substances were sought, those which act on homologous cells, and those which destroy foreign erythrocytes. The secretion by leucocytes of substances necessary to the nutrition of other cells was considered as probable long ago. Renaut thought that the main function of the white blood corpuscles was to bring to the fixed cells of the tissues the food material which they need. While the existence of physiological relations between leucocytes and tissue cells could be considered as almost certain, their nature had remained practically unknown. It was probable, however, that the substances secreted by leucocytes were analogous to the growth-activating and unstable substances which are found in embryonic tissues, leucocytes, and certain adult tissues. When connective tissue was aseptically inflamed, or when an aseptic peritoneal exudate contained many leucocytes, aqueous extracts of both connective tissue and peritoneal exudate were found to have acquired the power of stimulating cell proliferation. These experiments showed that leucocytes could bring to the tissues some activating substances. But it remained to be ascertained whether leucocytes, while they are alive, could secrete similar substances either spontaneously or under the stimulus of a foreign factor. Leucocytes are supposed to be, as is well know, the origin of the substances which protect the organism against infection. Although the problem of the origin of alexin and antibodies has been investigated by many experimenters, it is not yet completely solved. It was of interest, therefore, to ascertain whether leucocytic secretions could increase the natural hemolytic effect of hen serum on sheep or rabbit erythrocytes, and whether these secretions would become more active under the influence of a foreign protein. The substances which destroy foreign cells are not necessarily different from those which act on homologous cells. The word substance is used for simplicity of description and may be taken as meaning only a given property of an unknown substrate. A comparison was made of certain properties of sera extracted after 48 hours incubation from media containing leucocytes and from media containing no leucocytes. The serum from the leucocytic cultures was always found to be more favorable to the growth of homologous fibroblasts than the serum from the culture media incubated without leucocytes. The natural hemolytic power of the serum on sheep erythrocytes was found to be increased in about SO per cent of the experiments. In other experiments, we found that when two culture media free of cells were placed, one in an incubator at +38 degrees C. and the other in a refrigerator at +5 degrees C. for 48 hours, the serum from the incubated medium partly lost its hemolytic action on sheep or rabbit erythrocytes, while that from the refrigerated medium remained normal; at the same time, the inhibiting action of the incubated medium on homologous fibroblasts had increased very much. This effect of incubation indicates that certain unstable constituents of serum are destroyed by heat. Then the changes found in the properties of the serum from cultures of leucocytes are due to the fraction of the activating substances which has not been destroyed by incubation at 38 degrees C. A quantitative study of the secretions is, therefore, impossible with the present technique, which can furnish only qualitative indications about the substances set free by the leucocytes. We have ascertained also whether a medium containing leucocytes and kept in the refrigerator undergoes any change under the influence of the cells while they are in a condition of latent life. Gabritschewski dishes with and without leucocytes were placed in a refrigerator at a temperature of about +5 degrees C. After 48 hours, the hemolytic power on sheep erythrocytes of the serum from the leucocytic cultures had increased slightly and its inhibiting action on the growth of homologous fibroblasts had decreased. Then certain substances favorable to the growth of homologous cells and toxic for heterologous cells were diffused by the leucocytes into their medium. But the action of these substances was weaker than in the case of the cultures kept in the incubator. This experiment showed that leucocytes under certain conditions diffuse alexin or natural hemolysins which originate from them at the same time as the substances which activate homologous cells. In other experiments, although leucocytes were frozen at -10 degrees C., treated with distilled water, or extracted with saline solution, they did not yield any hemolysin. To summarize: Leucocytes, cultivated in plasma, always secreted substances which increased the rate of growth of homologous cells. Less frequently, they set free substances which hemolyzed foreign erythrocytes. The growth-promoting substances are analogous to those contained in embryonic tissues, and probably represent some of the foodstuffs brought to fixed tissue cells by leucocytes. They may possess the function of rejuvenating cells which have ceased to multiply when the cicatrization of a wound or the repair of a fracture requires a resumption of tissue activity. According to this hypothesis, the leucocytes brought to the surface of a wound by the process of inflammation would not only oppose bacterial invasion, but also bring to the tissues the material necessary to cell multiplication. It seems that in some cases regeneration is started by substances brought to the tissues by other cells. Loeb thinks that in Tubularia, when endodermic cells gather at the end where a new polyp is about to be formed, the substances given off by these cells are responsible for polyp formation.(6) There may be an analogy between this phenomenon and the secretion by leucocytes of growth-activating substances at the surface of a wound. If we assume that leucocytes in vivo set free their secretions in the blood stream, certain variations of the growth-inhibiting action of normal serum can be better understood. The rate of proliferation of homologous fibroblasts is much slower in the serum of an old chicken than in that of a young one. When the serum is heated at 56 degrees and 70 degrees C. for (1/2) hour, it becomes still more inhibiting. A substance favorable to cell activity has disappeared. It is therefore permissible to suppose that the growth-inhibiting power of serum and its variations are due to the antagonistic action of two substances, one growth-promoting and thermolabile, and the other growth-inhibiting and thermostable, the activating substance being always weaker in its effect than the inhibiting one. We know that activating substances can be extracted from embryonic tissue, from muscle and gland tissues, and from leucocytes of the adult animal, and that they are thermolabile and very unstable. Leucocytic secretions seem to have some of the properties of leucocytic extracts. It is probable that the activating substances which disappear from the heated serum are secreted by leucocytes and other cells. An increase of these secretions, then, would diminish the inhibiting action of serum on homologous fibroblasts. On the contrary, a decrease of the secretions in the serum would increase its inhibiting effect on homologous cells. The strong inhibiting action of serum in old age would be due partly to a reduction in the amount and activity of the substances secreted by leucocytes and tissue cells in the humors of the organism. Leucocytes also secreted in vitro substances which were toxic for foreign cells. Although the results were not constant, the serum appeared to become slightly more hemolytic for sheep or rabbit erythrocytes, under the influence of the leucocytes. The hemolysis of rabbit corpuscles by hen serum is due, according to Hyde,(9) to a complex sensitizer alexin, and not merely to alexin, as Bordet thought. When a foreign protein was added to the culture medium, the leucocytic secretions increased, as was shown by the action on homologous fibroblasts of sera taken from cultures of leucocytes with and without casein. The presence in the medium of the cultures of leucocytes of only 0.1 per 1,000 casein did not markedly modify the action of their serum on the proliferation of fibroblasts. When the concentration of casein in the leucocyte cultures reached 1 per 1,000, the growth of chicken fibroblasts in the serum extracted from the Gabritschewski dishes became more rapid. But there was no parallel increase of the hemolytic action of the serum upon sheep erythrocytes. We found that chicken serum containing 0.1 per 1,000 casein was barely toxic for homologous fibroblasts, while it became markedly inhibiting when the casein concentration reached 1 per 1,000. Probably, there is a relation between the toxicity of the medium, the increase of leucocytic secretions, and the time of the increase. The change brought about by casein in the equilibrium of the system composed of the cells and their medium determines the secretion by the leucocytes of substances which increase the activity of homologous cells and oppose the inhibiting effect of the foreign proteins. This reaction of the leucocytes is immediate, and may represent the first defense of the organism against a factor which disturbs its equilibrium. Possibly it differs from the specific cell reaction which leads to the production of antibodies. It is known that antibodies develop more slowly. Hemolysins were detected in cultures of bone marrow 4 days after the addition of antigen. The immunization of fibroblasts against foreign proteins has been shown by Fischer to begin after 4 days. If leucocytes behave in the organism as they do in vitro, we may assume that before the appearance of antibodies, they respond to the presence of an antigen by setting free growth-promoting substances and possibly alexin. This immediate reaction of the leucocytes against a disturbing factor, and the resulting production of substances which increase the activity of homologous cells, might be partly responsible for the results observed in the treatment of certain diseases by the injection of foreign proteins. It may be concluded that, under the conditions of the experiments: 1. The serum obtained from cultures of leucocytes is less inhibiting for homologous fibroblasts than the serum from media without leucocytes. In some experiments, its hemolytic action on sheep or rabbit erythrocytes is also increased. 2. The addition of casein to leucocytic cultures brings about a decrease in the inhibiting effect of the serum on homologous fibroblasts. 3. The increase in the activity of homologous fibroblasts in serum obtained from leucocytic cultures is probably due to growth-promoting substances secreted by the leucocytes. The presence of a foreign protein under certain conditions determines a more abundant leucocytic secretion.

Before proceeding to a discussion of the experiments upon cold-blooded animals, it is necessary to review briefly some of the work recently done with the bacillus of leprosy. The appearance of the bacillus in man and its behavior under artificial cultivation, and in the tissues of lower animals, should be considered in order that comparisons may be drawn. In their studies with the organism under cultivation, Duval and Gurd pointed out that the long, slender, and beaded appearance of the leprosy bacillus described by Hansen, in 1872, is lost when removed for several generations from the parent stem, and under artificial cultivation the organism becomes unbeaded, short, and coccoid. Duval also noted that these changes in morphology were always followed by rapid multiplication of the organism. Duval argues, a priori, that the bacillus is not in a favorable environment in the human tissues. If these deductions are correct, the morphology of the leprosy bacillus should vary according to the resistance offered by the tissues of different animals. The resistance of the human host to the leprosy bacillus becomes more evident in the light of the clinical aspect of the disease. The long period of incubation, the duration of the disease, and the disappearance of the bacilli preceding the healing of the infected foci show that the resistance offered to the bacillus by the human tissues is not to be overestimated. This opinion is confirmed when the behavior of the leprosy bacillus under cultivation and in the tissues of various mammals is compared. When cats, rabbits, bats, guinea pigs, and rats are inoculated either below the skin or into the peritoneal cavity with large quantities of Bacillus leprae, a slight local reaction follows within twenty-four to forty-eight hours, but no definite lesions are produced and the bacilli soon disappear. The resistance of some animals to Bacillus leprae is well illustrated by two cats which were inoculated subcutaneously and intraperitoneally with a heavy suspension of Bacillus leprae. These animals were killed and examined three days later, but the bacilli were not demonstrable from the regions about the sites of inoculation. Pigeons are likewise refractory. It is impossible to cause a local reaction in these birds, and the injected bacilli disappear rapidly. Hence, probably no multiplication takes place in them. Goats, young pigs, and white and dancing mice are in a degree susceptible to injections, and though undoubted lesions are produced, and multiplication of the bacilli occurs, the lesions and bacilli disappear after a limited time. Acid-fast bacilli which are recovered from the lesions are long, slim, and beaded, though the organisms used in the inoculations were short, unbeaded, and coccoid. Monkeys inoculated with cultures of the short unbeaded forms react promptly. The lesions resulting, though confined in most instances to the site of inoculation, occasionally appear at distant points. The number of bacilli present in the nodules and their arrangement within typical lepra cells show that multiplication has taken place. The organism has, however, changed from the short coccoid form to the long, slender, beaded form. Though the lesions induced and the bacilli present are in every way similar to those found in man, their tendency to disappear gradually after a quiescent stage clearly denotes that the tissues of the monkey, although less refractory than the tissues of the animals previously mentioned, still offer resistance to invasion. While mammals react but poorly to inoculations of the leprosy bacillus, this reaction manifests itself in various ways in different species. For example, while multiplication of the organism with the production of lesions occurs in some species, in others that are more refractory, the injected bacilli assume the involuted or beaded forms and do not multiply or produce lesions; in others, still more resistant to the action of the leprosy bacillus, the organisms quickly undergo granular metamorphosis and disappear. Furthermore, in some species the lesions are, in most instances, limited to the site of inoculation, and though presenting all the characteristics of the lesion in man, the nodules and the bacilli disappear after a variable time. This behavior of the leprosy bacillus can be accounted for only by the degree of resistance offered by the tissues of the individual host. Since the morphology of the organism invariably changes from the short coccoid to the large beaded form when placed in insusceptible animals, and conversely, from the long beaded forms to the short coccoid forms when placed in susceptible animals, the deduction can be drawn that the organism varies in morphology and rapidity of growth according to the susceptibility of the host. Examples of similar behavior of Bacillus leprae in the human subject are known to all investigators of leprosy. Ulcers and nodular areas often heal, and the bacilli disappear with little or no treatment. It is true that while older lesions are healing, new ones are constantly appearing, yet the duration of the disease and its undoubted tendency towards healing shows that conditions in the human subject are variable, and suggests that the organism has its natural habitat in some other host. The experiments presented here serve to show that the bacillus of leprosy meets but little or no resistance in the tissues of cold-blooded animals, multiplies in their tissues, and may be harbored by them without apparent discomfort or external evidence of the disease. That no appreciable resistance is offered to the multiplication of the leprosy bacillus by many species of cold-blooded animals is shown by the fact that aside from the trauma produced by the inoculation and the slight initial reaction of the tissues, the organism continues to grow profusely, and to invade the tissues without further reaction. Quite the opposite condition occurs in mammals: in some of these the leprosy bacillus degenerates into a granular mass shortly after inoculation; in others that are less refractory, typical lesions appear, but they seldom extend from the point of inoculation; and while the bacilli multiply slowly, they do not infiltrate the tissues, but disappear after a short time, the lesions healing. That multiplication of Bacillus leprae occurs in the tissues of cold-blooded animals is shown by the fact that while animals examined a few days after inoculation show but a few scattered organisms, those killed at longer intervals show a proportional increase in the number of bacilli. Furthermore, the few bacilli found at the early-period are extracellular and scattered, while after longer periods they tend to be massed and enclosed in large lepra cells. The supposition that these lepra cells are phagocytes has naturally arisen. Duval holds that they are not phagocytes in the true sense of the term, that the bacilli penetrate the cells rather than that the cells engulf them, after which, finding conditions for growth favorable, they multiply without causing serious injury to the cell. The size of the cell depends upon the size of the colony within. The experimental work bears out this view since the decrease in number of the organisms observed in animals killed shortly after inoculation depends not upon phagocytic action nor upon cells which appear later when active lesions are established. In early lesions, the lepra cells are smaller, barely measuring twenty to thirty microns in diameter, and contain but few bacilli; whereas in older ones, they attain a diameter of 100 microns or even more, and contain enormous numbers of bacilli. Were this increase in size due to phagocytic action, some cells would be found in which the limit of their capacity had been reached; and they would either contain a mass of dead and disintegrated bacteria or would themselves show evidence of disintegration. On the contrary, the bacilli, though they occupy most of the cell, show no signs of disintegration, and the nucleus and the cytoplasm of the cell retain normal staining properties. That the invasion and multiplication of the bacilli cause an irritation is evident by the amitotic divisions of the nucleus which occur in the larger cells. The absence of external evidence of invasion by Bacillus leprae in cold-blooded animals, and the apparent lack of discomfort caused by the presence of the organism within their tissues, are points which should be remembered in considering the sources from which leprosy may be transmitted. In not a single instance in the numerous experiments presented here would it have been possible, from any external sign, to suspect that the animals were harboring multitudes of leprosy bacilli. While the evidence in support of the opinion that leprosy may be transmitted from man to man appears sufficiently strong to warrant this belief, the number of cases in which infection can be actually traced to this source is small. Since leprosy is known to be prevalent where fish and sea-food are plentiful, and since the experiments here recorded prove that fish can be infected by being fed cultures of Bacillus leprae, or nodules from human lepers, or bits of fish previously infected with the leprosy organism, account should be taken of the possibility that leprosy, in certain localities, may arise from this source of infection. The question as to how and from what source leprosy bacilli enter the human body may be still regarded as an open one. Isolated examples of direct infection of healthy human beings from lepers have been reported by Arning and Nonne, by Manson, and others. The notion that the agency of infection is already infected human beings, that is lepers, is at the foundation of the modern practice of the isolation and segregation of lepers, which would seem to have brought about a definite decrease in the prevalence of the disease. It is an acknowledged fact, however, that the lepers confined in institutions practically never cause infection of nurses, etc. Some other factor than the human agency may therefore be considered as affecting this issue. It is well known that Jonathan Hutchinson has brought forward the idea that fish are the source of the infection, basing the view on the high prevalence of the disease along the coast countries of Norway and Sweden, and in the Pacific Islands, and in the countries bordering the Mediterranean and Black Seas, in all of which fish furnish the chief food material. No convincing proof was ever adduced in support of this contention. But now that it has been shown that the leprosy bacillus survives and multiplies in cold-blooded animals, at least at room temperature in a warm climate, and since methods have been devised for cultivating and identifying the leprosy bacillus, the question has been opened up to accurate investigation. Duval has shown that the leprosy bacillus in cultures grows better at room temperature than at 37 degrees C., so that growth in cold-blooded animals kept at room temperature is perhaps in some way connected with this phenomenon. What must now be ascertained, in order to test the Hutchinsonian theory more accurately is whether such growth takes place at a temperature corresponding with the average mean temperature of such a body of water as the North Sea and that of the fiords of Norway. Since cold-blooded animals possess the same temperature as their surroundings, they would be suitable media for the cultivation of leprosy bacilli at those temperatures. For the waters of the Mediterranean Sea and the tropical Pacific Ocean, this consideration would count less. But the theory will stand or fall according as it can account for the whole, and not only for a part, of the phenomena to be explained. As the length and shape of the bacilli and the number of chromatin masses are constant for a given species of cold- or warm-blooded animals, which features are governed by the resistance of the individual species, the following conclusions seem justified: that the morphology and rapid multiplication of the leprosy bacillus in cultures and in some species of cold-blooded animals indicate that Bacillus leprae under natural conditions is short, coccoid, and un-beaded, and that the long, slender, beaded variety which occurs in the mammalian species is atypical and the product of an unfavorable environment.

From the foregoing description of the histological changes in the leptomeninx it is quite evident that we are dealing with a chronic, stationary, healing form of tuberculous inflammation. This statement is substantiated, in the first place, by the clinical history. The only reasonable interpretation of the symptoms would establish the duration of the process as four months. The imaginable contingency that there existed first a meningeal syphilitic lesion that was dispersed by the iodide of potassium only to be followed by a tuberculous infection is so remote and unlikely that it need not be discussed. At all events the tuberculous leptomeningitis, which presented a typical distribution, began insidiously, existed at times in a latent condition, and pursued a very anomalous course, marked by a relative mildness of all the symptoms, and thus it came about that when an apparent or real improvement followed the administration of iodide of potassium able observers were induced to make an erroneous diagnosis. Death occurred as a result of an intercurrent infection. The long duration of the process is also shown, anatomically, by the thick layer of firm, translucent and gelatinous material that matted together the structures at the base, and also by the evident adhesions between the pia and the brain. The histological examination furnishes proof positive of the correctness of the conclusion in regard to the peculiar character of this process because it shows:(1) That the tuberculous proliferation is uniform in development and has reached nearly the same stage of evolution throughout the entire extent of the leptomeninx involved; it is not a process that has advanced by exacerbations and irregular extensions; the lesions are, generally speaking, of nearly the same age everywhere and must have begun at about the same time.(2) That only a very limited degree of caseous degeneration is present, pointing to an early arrest of the activity of the tubercle bacillus or to a very decided diminution or attenuation of its virulence.(3) That the subendothelial intimal proliferations of epithelioid cells, so generally found in acute tuberculous leptomeningitis,* have in this case become more or less completely changed into distinct fibrous tissue in which but very slight, if any, direct evidence of its tuberculous origin can be found. It is only by recognizing that the chronic endarteritis is most marked in correspondence with the most advanced adventitial tuberculous changes, and by finding an imperfect, much altered giant cell in one district of intimal thickening, that we were able to establish the direct kinship of the endovascular changes with those of the pia in general.(4) That acute inflammatory changes, in the form of emigration of polymorphonuclear leucocytes and of fibrinous exudation, are entirely absent in all parts of the district involved. The presence of a turbid serous fluid is of course not at all inconsistent with the view that the anatomical changes are of long duration.(5) That the granulation tissue present is, in general, undergoing fibrillation and contains a rich supply of enabryonal capillary vessels as well as of larger blood-vessels of evidently new formation. The absence of any considerable extent of polymorphonuclear leucocytic infiltration in this tissue has already been referred to. The cells in the granulation tissue correspond to the cells of embryonal or formative connective tissue. Vacuolation is rarely present.(6) That the unusually large number of giant cells present are remarkably free from evidences of necrosis and degeneration of the character ordinarily observed in tuberculous proliferations, that they do not contain in demonstrable form tubercle bacilli, and that the majority of the giant cells seem to be separating into individual cells and smaller masses often with, but sometimes also without, evidences of nuclear disintegration. The possibility that these phenomena may signify fusion instead of the sundering of cells will be discussed below. For these reasons there can be no doubt that the general claim that we are dealing with an instance of chronic, healing tuberculous meningitis must be regarded as established beyond dispute. The growth of tubercle bacilli in the glycerine-agar tubes, inoculated with the fluid from the pial meshes, and the demonstration of tubercle bacilli, though in very small numbers, between the cells of the embryonal tissue, furnish the positive evidence that we are actually dealing with a tuberculous process due to living and not to dead bacilli. The degree of virulence of the cultures of tubercle bacilli was, unfortunately perhaps, not studied. The presence of living tubercle bacilli in a tissue free from active and acute changes characteristic of tuberculosis demonstrates that, whatever the actual degree of virulence of the bacilli may have been, the tissue in which they were found was at this time relatively immune from their action. The manner in which this immunity was produced, and in which the process of healing was initiated, need not be discussed at this time any further than to again direct attention to the fact that the bacilli lost their virulency as regards the cells in this leptomeninx before these cells underwent any marked degree of degeneration. The cells of the tuberculous proliferations survived the further action of the bacilli whose original effect it was to initiate cell accumulation or proliferation; the cells also retained sufficient vitality to develop, in some instances at any rate, into formative cells according as their origin would dictate, e. g. into fibroblasts. That fibroblasts are formed only by embryonal connective tissue cells, and not by wandering cells, such as the large mononuclear leucocytes, we are well aware, is possibly still a disputable assumption, and we do not consider it pertinent to discuss the question any further in connection with this study, but would only emphasize the point that some of the cells of tuberculous proliferations may, under favorable circumstances, become formative cells, and, furthermore, that the amount of formative tissue produced may be far in excess of what is actually needed for purposes of repair only. Surely the appearances here noted indicate that the bacillus of tuberculosis has the power to stimulate fixed cells to multiply, unless one assumes that all, or almost all, the formative cells here seen are derived from wandering cells attracted by the presence of the bacillus and its products. As to the ultimate fate of the formative and other cells in this healing tuberculous tissue no final statements can be made. It must be remembered that it is only one stage in the process of healing that is dealt with. The well marked evidences of fibrillation, the quite extensive formation of new vessels, the absence of evidences of degenerative changes in the uninuclear cells, all point to the production of new fibrous tissue as sure to occur, but it seems quite probable that occasional epithelioid cells may undergo or have undergone dropsical or other forms of degeneration, although it is certainly apparent that so far as the small cells are concerned the involution of the tuberculous tissue is not occurring through disintegration. Perhaps the most interesting feature in this case is the opportunity it affords to study the changes in the giant cells of healing, non-degenerated tuberculous tissue. In the first place, the large number of giant cells is quite remarkable. The general characters of the tissue in which they are found recall the fact that giant cells are regarded as quite constant elements in chronic mild tuberculosis; often the giant cells are the only cells that contain bacilli (Koch). In this instance the giant cells do not contain bacilli that are demonstrable by the usual methods; neither do they contain bodies that can be definitely interpreted as degenerate forms of bacilli such as those found by Metchnikoff, Stchastny, Weicker, and others, in the giant cells of Spermophilus guttatus, in avian and in human tuberculosis. Metchnikoff states, however, that he knows of the occurrence of such degenerate forms only in the Spermophilus guttatus under the circumstances mentioned, and in the rabbit and guinea-pig in mammalian tuberculosis, but not in man; consequently, the manner in which the giant cells rid themselves of the bacilli undoubtedly present in their interior at some time during their existence, must as yet remain without any explanation. In the description of the histological changes the various appearances presented by the giant cells are described somewhat minutely. The essential observations made concern, in my opinion, the further fate of giant cells which are still found to persist in healing nondegenerated tuberculous tissue. It was, I believe, quite conclusively shown that the consecutive changes appear to consist in the breaking up of the nuclei, the removal of the detritus by phagocytes, and the formation of a few apparently viable uninuclear cells in the case of more degenerated, exhausted giant cells, while other, and, as it would seem, better preserved or younger giant cells, separate into a number of individual, uninuclear cells with but little or no nuclear disintegration. Objection might be raised to this interpretation of the appearances in the giant cells. While no one could very well dispute the view that part of the giant cells are undergoing retrogressive and absorptive changes with the production of some viable cells, a question might well be raised concerning the nature of the process taking place in those giant cells that have been spoken of as splitting up or dividing into uninuclear cells and smaller multinucleated masses without much evidence of nuclear disintegration. It might be claimed that the process is one of fusion of many cells to form giant cells, and not one of division of fully formed giant cells into small cells. But a broad view of the processes described speaks against fusion. In the first place we are not dealing with a stage of tuberculous proliferation (Baumgarten), or cell accumulation (Metchnikoff), in which one would look for the production of giant cells, no matter which view concerning the histogenesis of tubercle be assumed as the correct one, because it has been demonstrated that, from whichever point of view the lesions are examined, the same positive conclusion that they are in the process of healing is reached; there is, therefore, no occasion for the formation of new giant cells in such wide-spread degree throughout the district involved. It might he claimed that the cells became arrested and, as it were, fixed in the act of fusion which was taking place in the early stage of the meningitis, but it would be difficult to understand the nature of the stimulus that could hold the cells together in such a peculiar manner for such a long time. It must be remembered that bacilli or bacillary detritus could not be found among the incomplete or in the complete giant cells. In the second place the difference between the cells that are undergoing disintegration and those regarded as dividing is essentially, to a certain extent at any rate, one of degree, because in the first instance there is not much, if any, doubt but that viable smaller cells are also formed, and in the second instance some, though often very slight, evidence of nuclear fragmentation is nearly always present; it would also be correct to infer that in advanced subdivision of a giant cell much, and perhaps all, of the nuclear detritus produced might have been removed up to the last trace; finally, the two extremes of these changes in the giant cells are connected by transition stages passing by gradation from the one to the other. Hence it is justifiable to conclude, for the time being, that in healing non-degenerated tuberculous tissue, the multinucleated giant cells may in part disintegrate and undergo absorption, in part form viable small cells; that both these changes may, and usually do, affect the same cell, but that in one class of cells-presumably the older or the more exhausted-the retrogressive process is predominant, while in a second class of cells-presumably the young and vigorous-the progressive changes are the more marked. In this connection it may be pointed out that while there cannot very well be any question but that we are dealing only with dividing and not coalescing cells, yet if this conclusion should be disputed and found incorrect, then the only remaining alternative would be to infer that this tissue furnished a unique and striking example of the formation of plasmodial masses by fusion in human tuberculosis, a conclusion to which many pathologists would refuse to subscribe, if for no other reason than because it is not in accordance with the almost universally accepted teachings of Baumgarten and Weigert in regard to the mode of formation of the giant cells in tuberculosis. Believing as I do that the giant cells under consideration are in the act of division and not at all of fusion, there remain to be discussed some of the histological and other features presented by the dividing cells. Many of the giant cells, perhaps the majority, contain larger and smaller vacuoles in the protoplasm. The exact significance of this vacuolation is not always clear. When the vacuolation accompanies an evident solution of the nucleus (karyolysis), there cannot be any doubt but that we are in the presence of a distinctly retrogressive process. Vacuoles are also most numerous in the giant cells that present other evidences of degeneration, such as coarseness of the granules in the protoplasm and extensive nuclear disintegration, but they occur as well around nuclei that stain deeply, around cells that seem to be separating from the giant cell, and even about nuclei that present mitoses. The formation of vacuoles seems to be responsible, to a certain extent at any rate, for the diminution in the volume of disintegrating and dividing giant cells, as shown by the clear spaces that form about them; these spaces are too large and occur too uniformly to be attributed solely to artificial shrinking produced by the hardening in alcohol. Further undoubted evidence of retrogression in certain giant cells is the occurrence of nuclear disintegration, or karyorhexis, which sets free larger and smaller chromatin masses that are recognized in the giant cell as well as in the interior of the phagocytes usually found around such cells. Almost all the polymorphonuclear leucocytes found in this tissue are met with around giant cells with broken-up nuclei. In many nuclei of disintegrating giant cells can be noted appearances that correspond well to certain stages in the complicated karyorhexis observed in anaemic necrosis by Schmaus and Albrecht; some of the nuclei with budding processes correspond particularly well with those in certain of their drawings; the interior of giant cells of tuberculous tissue may, it would seem, present conditions favorable to the development of this series of postnecrotic nuclear change. Vacuolation, karyolysis and karyorhexis are the essential steps that lead to destruction of the whole or parts of some of the giant cells; associated with these processes there is usually observed a splitting up of the body of the giant cell into irregular fragments with as well as without nuclei; and, as described, more or less phagocytosis of the resulting remnants of various kinds is seen. But evident degenerative and necrotic processes in a giant cell may be associated with progressive changes. While some nuclei undergo vacuolation or break up, others seem to become richer in chromatin and to stain more deeply at the same time that they seem to acquire cell bodies quite distinct from the protoplasm of the giant cells: this hyperchromatosis does not, therefore, seem to be a stage in karyorhexis. A very few but undoubted karyokinetic figures were found, together with evidences of division of the cell body formed in the giant cell protoplasm. Precisely similar changes are described by Klebs in healing pulmonary tuberculosis of the guinea-pig; the nuclei of the giant cells became rich in chromatin and karyokinetic figures occurred. Krückmann among others has found occasional mitoses in giant cells around foreign bodies, as well as elsewhere, but it would seem that such mitoses have always been interpreted as indicating the probable mode of formation of the giant cells rather than of their involution. The question of mitosis in existing multinucleated cells has recently been studied by Krompecher, who concludes that the individual nuclei of such cells may undoubtedly divide by mitosis, either simultaneously or at separate times. Division by amitosis can also occur, but mitosis is the only progressive form of division, amitosis being a retrogressive, disintegrating process that must be looked upon as an evidence of degeneration of the nucleus. Ziegler states that in division of giant cells whose nuclei have multiplied by mitosis it may happen that the separating cell remains enclosed in the protoplasm of the mother cell. A singular phase in the involution of the giant cells in this pia is to be found in the existence of progressive changes side by side with nuclear necrosis and with degeneration; this finding indicates that giant cells may contain many independent elements which, though apparently fused into one large cell, may preserve their individuality so that while some nuclei die, others proliferate and perhaps feed on the remnants of their dead brethren and form new, viable small cells. The nuclei in giant cells may be looked upon as representing independent centres, capable at times of existing even though the cell protoplasm is disintegrated. Many of the giant cells separate into individual cells, unaccompanied or unassociated with much evidence of necrosis. These cells may be regarded as the more vigorous forms. Here also are observed occasional mitoses-but on the whole extremely few-and very constantly an evident increase in the amount of chromatin in the nuclei of the new cells as compared with the amount ordinarily found in the nuclei of giant cells. These deductions concerning the persistence of the vitality of some of the nuclei, even in the presence of molecular and morphological changes in the cytoplasm and in other nuclei of the giant cell that lead to disintegration, are not entirely without the support of previous observations on cells, which, although made under different conditions, are nevertheless, it would seem, applicable to cells in general. Thus the brilliant investigations of Loeb upon the effects of various unfavorable surroundings, such as absence of oxygen or reduction of the amount of water, upon the cleavage of eggs of many kinds, show that the conditions which arrest development are qualitatively alike for nucleus and protoplasm, but quantitatively less for the protoplasm; when the irritability of the protoplasm is suspended the nucleus may segment without segmentation of the protoplasm, but upon re-establishment of favorable conditions the protoplasm may divide into about as many spheres as there are nuclei preformed-the nucleus persists, preserves the irritability of the cell and stimulates the protoplasm to segmentation. From the appearances of the giant cells here described it would seem, then, that some nuclei are able to maintain their vitality longer than others in the same cell, and under certain conditions to stimulate parts of the protoplasm to segment; in other cells all the nuclei have, as a rule, preserved their irritability. The groups of cells formed by the dividing of the giant cells can be traced by studying the process at the different stages in the different parts of the tissue. They assume an oval or spindle-shaped form, becoming more and more like the formative and endothelioid cells of young connective tissue, but their ultimate fate cannot be determined because it concerns essentially only one limited period in the involution of the tissue. It may be said with reasonable certainty, however, that the new cells do not form blood-vessels, but as regards their forming lymph-vessels nothing definite can be concluded. It would not be safe to draw any definite conclusions, from the appearances described, with regard to the origin and the mode of formation of the giant cells. The resulting small cells in general resemble very much endothelial and formative cells, but some of them are, at certain stages at any rate, not unlike large mononuclear leucocytes; their final fully developed or mature condition being unknown, no positive inference can be drawn as to their pre-giant-cell origin. The evidence points to the fact that the most probable origin of the giant cells, as indicated by their form and the apparent future career of their descendants, would be the fixed mesoblastic cells of the pia. In regard to the mode of formation of the giant cells it is quite clear that it must involve some process which is not incompatible with the viability of the small cells which may spring from the giant cells. Whether this would speak more in favor of formation by fusion than by karyokinesis of a single cell without division of the cell body cannot be well determined, and as long as authors are not agreed upon the question of the production of living, procreative cells by amitosis (direct segmentation, direct and indirect fragmentation) it would not be profitable to discuss the compatibility or incompatibility of the views of those investigators who trace the origin of giant cells to amitotic division, with the progressive changes that giant cells have been shown to be capable of. The fact that giant cells in tuberculous tissue, under certain conditions, undergo progressive changes and separate into small, living cells proves that they are not, as claimed by Baumgarten, Weigert and others, necrobiotic elements that are doomed to destruction from their very inception. On the other hand it lends more strength, if that were necessary, to the teleological view urged by Metchnikoff that they are living, defensive cells (whatever their origin may be), formed for the distinct purpose, like plasmodial masses in general, of isolating and removing foreign, harmful bodies, in this case the tubercle bacillus, and, having accomplished their object without being destroyed or exhausted, or the cause of their formation being removed or neutralized in some way, they, or their nuclei, may retain enough irritability to form a larger or smaller number of living, small, uninuclear cells.

Abstract Background Colonic hydrotherapy is widely used and many of its practitioners are medically qualified. Nonetheless, the basis of many of their practices requires physiological scrutiny. Method The claims of colonic hydrotherapy are examined against known physiological facts. Results Hydrotherapy is not entirely physiological. Conclusion Colonic hydrotherapy may increase the dissemination and absorption of toxins and bacteria into the body.

Sympathetic nerve activation is recognized at the adipose tissue level during cold exposure. Adiponectin is a key protein produced by adipose tissue, but its acute modulation remains unknown in humans exposed to cold. The aim of this study were (1) to examine the acute effects of cold exposure on circulating adiponectin and (2) to determine whether the changes are modulated by (a) an acute glucose ingestion as well as (b) a short-term modulation in carbohydrate (CHO) availability. Using a random crossover design, 6 healthy men were exposed to cold for 120 minutes with ingestion of beverages containing low (Control, 0.04 g/min) or high (High, 0.8 g/min) amounts of glucose during the course of the experiment (study 1). In study 2, 6 healthy men were exposed twice to cold for 120 minutes after equicaloric low-CHO diet and exercise and high-CHO diet without exercise. Plasma adiponectin concentrations were quantified before and during cold exposure. In study 1, adiponectin levels did not change during High, whereas a 20% rise was observed during Control (condition x time interaction, P =.06). In study 2, adiponectin levels increased by approximately 70% during cold exposure after both low- and high-CHO diets (effect of time, P <.05). A 120-minute period of cold exposure is accompanied by a significant increase in adiponectin levels in young healthy men. The rise in adiponectin levels observed during shivering is inhibited with glucose ingestion but not after diets varying in CHO content.

OBJECTIVE: Depression is more than just a mood disorder, it is a real illness that not only affects one's mood and thoughts but also appetite, sleep patterns and one's self esteem. Today by primary care physician every fourth patient is diagnosed with depressive disorder where 15% of them try or commit suicide. Objective is to correlate importance, frequency,& recognizing physical symptoms who indicate depressive disorder. METHODS: Research pooled sample of 33 female patients who seek medical attention with numerous physical pains which had no organic cause (n = 33). Participant's average age was 46. They were mainly unemployed and single mothers with one or more children. Seven physical symptoms were observed before, during diagnosis and their existence after two months of anti-depression therapy. They are: a) headaches; b) rapid heartbeat; c) dizziness or hightheadedness; d) shortness of breath; e) increased sweating; f) stomach aches; g) nausea. Research took place between June and December 2007 in the office of family medical practice. Data was collected & analyzed using program SPSS14. RESULTS: In the beginning of disorder, the physical symptoms are milder and increasing with time and development of disorder. The most common physical symptoms, headaches, rapid heartbeat, dizziness or lightheadedness, and nausea are present before and after anti-depressive or anxiolytic therapies. Statistical analysis differentiating number of symptoms before and after therapy shows 5% decrease. After two months of treatment physical symptoms, headaches, rapid heartbeat, and nausea remain where dizziness or lightheadedness, stomach aches, increased sweating or breath-shortness largely respond to therapy. CONCLUSION: The research shows physical symptoms take significant place in recognizing depressive disorder. They are accompanying symptoms of depressive disorder. Most common physical symptoms of depressive disorder are: headaches, rapid heartbeat, dizziness or lightheadedness, and nausea. There is significant statistical difference in the number of physical symptoms before and after anti-depressive and anxiolytic therapies.

One of the first neurobiological theories of major depression was the monoamine deficiency hypothesis. The classic monoamine theory of depression suggested that a deficit in monoamine neurotransmitters in the synaptic cleft was the main and primary cause of depression. Recent and newer versions and modifications of the primary classic theory also mainly included this postulate, while other theories of depression preferred departing from the monoamine-based model altogether. Unfortunately, the clear neurobiology of major depression remains an elusive issue, despite intense research. It is clearly held that most, if not all, antidepressant pharmacotherapies treatments produce their therapeutic antidepressant effects, at least in part, by modulating monoamine systems (noradrenergic, serotonergic, and dopaminergic) by a selective or a multi-acting way; however, much less is known about the neurobiological pathology of these monoamine systems in depression. Much of the past 10-15 years of research in the biology of mood disorders has led to considerable evidence in depression implicating multiple system pathology, including abnormalities of monoamine as well as other neurotransmitter systems. These approaches and findings have led researchers to propose broader theories regarding the neurobiology of depression, just like a spreading disorder of specific neuronal networks in the brain. A model for the pathophysiology of depression ill be discussed in the next pages, after describing the main components of depression pathogenesis. Suggestion is that the primary defect emerges in the cross-regulation and vulnerability of special monoaminergic and non-monoaminergic neural networks, which leads to a decrease in the tonic release of neurotransmitters in their projection areas, altering postsynaptic sensitivity, and following, overexaggerated responses to acute increases in the presynaptic firing rate and transmitter release. It is proposed that the primary defect should be involved, in the noradrenergic innervation spreading from the locus coeruleus (LC). Dysregulation of the LC projection activities may lead in turn to malfunction of serotonergic and dopaminergic neurotransmission. Failure of the LC function could explain the basic impairments in the processing of novel information, intensive processing of irrational beliefs, and anxiety. Consecutive deficits in the serotonergic neurotransmission may contribute to the mood changes and reduction in the mesotelencephalic dopaminergic activity to loss of motivation, and anhedonia. Malfunction and dysregulation of CRF and other neuropeptides such as neuropeptide Y, galanin and substance P may reinforce the LC dysfunction and thus further weaken the adaptive ability to stressful stimuli. The new SNRI antidepressants seem to be more superior and effective in the treatment of major depression and in the prophylaxis of recurrent depressive episodes because of their coexistent noradrenergic activity.