Success of cell replacement therapies for neurological disorders will depend largely on the optimization of strategies to enhance viability and control the developmental fate of stem cells after transplantation. Once transplanted, ste/progenitor cells display a tendency to maintain an undifferentiated phenotype or differentiate into inappropriate cell types. Gain and loss of function experiments have revealed key transcription factors which drive differentiation of immature ste/progenitor cells towards more mature stages and eventually to full differentiation. An attractive course of action to promote survival and direct the differentiation of transplanted stem cells to a specific cell type would therefore be to force expression of regulatory differentiation molecules in already transplanted stem cells, using inducible gene- expression systems which can be controlled from the outside. Here we explore this hypothesis by employing a tetracycline gene regulating system (Tet-On) to drive the differentiation of boundary cap neural crest stem cells (bNCSCs) toward a sensory neuron fate after transplantation. We induced the expression of the key transcription factor Runx1 in Sox10-expressing bNCSCs. Forced expression of Runx1 strongly increased transplant survival in the enriched neurotrophic environment of the DRG cavity, and was sufficient to guide differentiation of bNCSCs towards a non-peptidergic nociceptive sensory neuron phenotype both in vitro and in vivo after transplantation. These findings suggest that exogenous activation of transcription factors expression after transplantation in ste/progenitor cell grafts can be a constructive approach to control their survival as well as their differentiation to the desired type of cell and that the Tet-system is a useful tool to achieve this.

1. A virus capable of producing pulmonary consolidation in the wild mongoose (Herpestes griseus) has been isolated from throat washings obtained from four patients with a clinical syndrome termed acute pneumonitis. 2. The virus was not pathogenic for ferrets, mice, guinea pigs, rabbits, monkeys, voles, hamsters, deer mice, skunks, opossums, or woodchucks. 3. The virus was filterable through Berkefeld V and N candles, was not inactivated by glycerin or by freezing and drying in vacuum, and was propagated for at least 30 serial passages on the chorio-allantoic membrane of the developing chick embryo. 4. Normal mongooses placed in contact with infected mongooses developed pulmonary consolidation. 5. The virus was neutralized by the serum of mongooses convalescent from the infection but was not neutralized by normal mongoose serum. 6. Serum of human beings convalescent from acute pneumonitis also neutralized the virus, but serum obtained from the same individuals during the acute phase of the disease failed to do so. 7. The evidence so far obtained strongly suggests that this virus is the cause of acute pneumonitis in human beings. It differs from other viruses known to cause infections of the respiratory tract in man.

Five strains of virus were recovered from nasal washings and feces. Four strains were of human origin, the fifth strain came from a monkey sacrificed at the height of the disease. Of the four human strains the first was isolated from the feces of a 14 year old child 7 days after the onset of illness. The second strain was from the nasal washings of a 6(1/2) year old child, 5 days after the onset of illness. The third and fourth strains were recovered from the same patient, a 2(1/2) year old child, 9 days after the onset of illness. One of these strains was obtained from nasopharyngeal washings and the other from the feces. The single monkey strain was isolated from the upper intestinal segment and appears to be the only instance of its isolation from this source in the literature. We believe that the detection of the virus in the nasal washings of two additional patients during convalescence lends further support to the belief that the virus of poliomyelitis is spread by human contact. Furthermore, the recovery of the virus from the gastro-intestinal tract with as great or greater frequency as from the upper respiratory tract, need not, it appears to us, alter our concept of the mode of entrance of the virus into the body, namely, by way of the upper respiratory tract. If the presence of the virus is conceded, then a consideration of the physiologic passage of nasal and oral secretions into the gastro-intestinal tract by reflex swallowing would serve to explain adequately the presence of the virus in those organs. It might even be further predicated that since the gastro-intestinal tract functions as a temporary reservoir for secretions from the upper respiratory tract, the gut should, after a time, contain the virus in higher concentration than any single sample of secretion obtained from the upper respiratory tract by nasal washing. It appears to us that failures to detect the virus in the gastro-intestinal tract are perhaps more indicative of inadequate procedures for its detection than of its absence. The recovery of the virus from the feces 7 and 9 days after the onset of illness takes on added significance. It indicates first, that the virus withstands the gastric acidity which under normal physiological conditions tends to keep gastric contents relatively free of bacteria. It further suggests that improper disposal of feces from patients with poliomyelitis may have serious public health consequences, particularly in smaller communities where inadequate sewage disposal may result in contamination of surrounding beaches or even local water systems.

Pneumococci, H. influenzae, and S. hemolyticus are known to be frequent inhabitants of the upper respiratory tract, but most workers have not recognized any definite relationships between their presence and coryza, sore throat, influenzal, and sinusitis attacks (2-5). Dochez, Shibley, and Mills, however, in their experimental studies of common cold, state that in both the spontaneous and experimentally induced "colds" in anthropoid apes, the "most significant change observed has been the increase of activity on the part of the potential pathogens habitually present in the throat flora. Coincident with the appearance of symptoms, pneumococci, S. hemolyticus, and B. pfeifferi have developed in greatly increased numbers and have spread over a wide area of the nasopharyngeal mucous membranes. These organisms became at this time conspicuous even in the nose, where they are seldom or never present under normal conditions. The same phenomena have not been observed in human beings"(6, 7). The essential facts of the present observations are that persons free of pneumococci, H. influenzae, and S. hemolyticus are in general free of coryza, sore throat, influenzal and sinus attacks; that persons who are occasional or periodic carriers of these organisms may be negative on tests over long healthy periods, but generally become positive during or following attacks and subsequently become negative again; finally, that persons who are chronic carriers show during these illnesses increasing numbers of organisms in the throat and extension of the organisms to the nose. That these organisms may be the actual incitants has been claimed by Park (8); that they are secondary invaders is the view of Shibley, Mills, and Dochez who state as a result of their experimental work on this subject that "the most important significance of viruses of this type [common cold] seems to lie in their capacity to incite activity on the part of the more dangerous pathogenic organisms that infect the upper respiratory tract"(7). The present observations bring out the intimate relationship between these pathogens and upper respiratory tract symptoms, but do not disclose the nature of this relationship. Finally, an addition has been made to the knowledge of the mode of spread of these organisms. A focus of growth and dissemination has been determined in the nasal passages and throat of individuals with chronic upper respiratory tract disease and increases in numbers of the organisms at the focus and their spread to contacts have been related to the winter season and to the occurrence of symptoms in the carrier. The observations suggest that the dosage of these organisms in a community is controlled by the resistance of the carrier and of the contacts. This view is in agreement with the facts derived from studies of native animal infections (9).

Experimental upper respiratory infections similar to "common colds" were transmitted singly and in series through two and four passages in nine out of fifteen persons, by intransal inoculations with bacteria-free filtrates of nasopharyngeal washings obtained from individuals ill with natural "colds." These observations conform with those reported by previous workers and lend further support to the view that the incitant of the "common cold" is a filtrable virus.

In our experience, covering a large number of rabbits, we have found that the condition known as snuffles falls into different types, the acute and fatal, symptomatic of some underlying infection such as septicemia or pneumonia; and the intermittent, and the chronic. The intermittent and the chronic types considered in this paper are those most commonly present in laboratory stocks. Our observations point to a widespread prevalence of the disease among rabbits kept under laboratory conditions. This statement might be questioned had the stock we examined been derived from a single source. But the animals were procured from dealers who obtain rabbits in New York, Pennsylvania, Ohio, and as far west as Michigan. The same dealers supply the stock of most of the laboratories in the East. Hence we believe that this disease is found generally prevalent in this part of the country. We have demonstrated that practically all rabbits with intermittent or chronic snuffles reveal old chronic inflammatory processes of the upper nasal passages associated with thick inspissated pus in one or more sinuses. Moreover, these conditions are also present in about one-tenth of carefully selected, supposedly normal stock rabbits and in one-fourth of a casually selected group, free, during long periods of observation, from any of the symptoms of active snuffles. The peculiar anatomy of the animal's nose which predisposes to the ready formation of enclosed pockets of purulent material may be the cause for the chronicity of upper nasal affections. When the animal's resistance is lowered, the long standing, inflammatory process can flare up into an acute exacerbation, and then show itself as typical snuffles. Various means can effect this: chilling the body, the intravenous injection of foreign proteins such as killed vaccines, or the intranasal inoculation of microorganisms of divers species. Bacteriological examination of the nasal secretions or sinus pus from animals with snuffles and those apparently free of the disease shows the presence in both cases of various microorganisms-Staphylococcus albus, Bacillus bronchisepticus, Bacillus lepisepticus, and others, in order of frequency. Different bacteria may be found in different sinuses in the same animal. A lack of recognition of these factors has led, we believe, to erroneous conclusions with regard to the inciting agent of the disease. Bacillus lepisepticus and Bacillus bronchisepticus have been declared the incitants of snuffles. Our experiments, in which an attempt was made to induce the disease de novo with these microorganisms, failed. In all cases (with a single exception) in which snuffles followed, there was evidence of an infection which, judging from the condition of the nasal passages and from the cells in the exudates or secretions, had existed before the inoculations were made. Furthermore, the microorganisms recovered from the nasal passages had as a rule no relationship to those in the material inoculated. We attempted also to produce snuffles by inoculating intranasally the unfiltered and filtered suspensions of the ground nasal mucous membranes from typical cases of the disease occurring in stock rabbits. These attempts also failed. It appears, therefore, that intermittent and chronic snuffles, as it attacks rabbits kept under laboratory conditions, is, as a rule, a sign of an underlying condition-an exacerbation of a chronic inflammatory process in the upper nasal passages, associated with a purulent paranasal sinusitis. The microorganisms recovered are to be looked upon as tending to maintain such conditions but we have still been unable to reproduce typical snuffles with them, employing supposedly normal stock rabbits for the purpose. One may presume that some agent, as yet undetermined, diminishes the resistance of the nasal mucosa, allowing different bacteria to invade and multiply there, thus causing disturbance. In this respect perhaps an analogous condition exists to that which prevails in epidemic influenza and common colds in man. It is obvious that further work along these lines cannot be properly carried out with rabbits whose antecedent history is unknown. The problem of the incitant of snuffles can best be studied in a breeding stock which is well controlled, one affording an opportunity to observe the animals from an early period of life.

From the foregoing experiments it appears that with the filtered nasopharyngeal secretions from early cases of typical infections common colds in the first 3 to 18 hours of the disease, a similar condition can be transmitted to man. With the unheated but not with the heated secretions from four of six such patients we have succeeded in transmitting an affection indistinguishable from common cold to four men and in two instances the condition was conveyed from the person with the experimental disease to a second individual -in all, therefore, to six supposedly normal subjects. The periods of incubation in the experimental disease varied from 8 to 48 hours. We failed to obtain these results with the filtered secretions from cases of common colds 18 and 20 hours after the onset of symptoms and from a patient with the experimental disease 20 hours after the first symptoms. It would appear that the secretions are more active in the early hours of the affection. We also failed in the two instances in which colds were caused by exposure to the elements, or chilling of the body, and not by definite contact with other cases of common colds. Intratracheal inoculations in rabbits with unfiltered and filtered nasopharyngeal washings obtained from patients with common colds induce no characteristic or distinctive effects on the tissues, from which no constant, pathogenic agent has, as yet, been recovered. In comparison, similar material from cases of epidemic influenza do, however, cause particular changes in the blood and lungs of these animals, cultures of the lungs often yielding Bacterium pneumosintes. In view of these facts and since the clinical pictures exhibited by these diseases differ, the conclusion may be drawn that infectious common colds and epidemic influenza are separate and distinct diseases. On the other hand, the negative results obtained with materials derived from common colds and from parallel series of experiments with secretions from supposedly healthy persons, serve as a control to the effects produced with the nasopharyngeal washings obtained from influenza patients. Aerobic and anaerobic cultures of the filtered nasopharyngeal washings from 40 early cases of infectious common colds have thus far yielded no constant, pathogenic agent which can be regarded as the incitant of the disease. The filtered washings of nineteen cases were studied by the combined method of Smith-Noguchi fluid medium and anaerobic blood agar plates. In these instances representatives of the three groups of anaerobic filter-passing, Gram-negative bacteria, described by Olitsky and Gates were cultured from twelve patients. The irregularity of their occurrence not only in common colds but in influenza and supposedly normal persons and their lack of pathogenicity for rabbits and man indicate that these bacteria are not peculiar to common colds. This method has opened to view a number of hitherto undescribed microorganisms which can be found in different respiratory affections and in health. Furthermore, by morphological, cultural, and serological means, the separation into distinct species of each of these groups of bacteria has again been demonstrated. It is noteworthy that Bacterium pneumosintes was not found in any of the cultures from the 40 patients. Special attention was given to the detection of elements similar to Foster's globoid bodies in the cultures derived from common colds and from the experimental disease in man, and from the lungs of inoculated rabbits. We have not been able to determine the presence of these bodies, although the precipitate which forms in fresh rabbit kidney tissue-ascitic fluid medium was illusory in such relation since it was a common experience to find this precipitate simulating the globoid bodies of poliomyelitis. Still more disturbing is the fact, that these particles could be carried over from subplant to subplant and even showed pseudo colony formation in the Noguchi semisolid medium in tubes. But when the particles were put to rigorous test for a living and multiplying organism, the tests failed to reveal multiplication. The increasing importance of the tissue-ascitic fluid medium in bacteriological technique warrants a detailed description of the requirements necessary for the determination of the living nature of formed elements in cultures in this medium,(a) No one method of staining can be relied upon, for stain precipitate in itself adds to the confusion. A specimen for examination should be stained separately by Gram's and Giemsa's methods and with another nudear dye, such as polychrome methylene blue. As a rule, microorganisms will reveal their morphological characters in more than one stain, whereas precipitate may be found in only one and not in the others. The experienced eye will discern the precipitated particles in selected parts of the stained preparation where they often occur in enormous numbers, clumped into irregular masses of varying forms from the periphery of which there is a gradual fading out to finer, more uniform structures.(b) In addition, suspected growths should be tested in the dialysate medium of Gates since by this method the precipitated material of the medium is kept from admixing with the growth, and a clear view of any microorganisms, if present, is obtained,(c) Another requirement is colony formation of the suspected culture. This is an absolute essential and can be effected by planting the material to be tested on solid plate media, incubated aerobically, and anaerobically in Brown's jar. Semisolid medium in long tubes should also be employed but care is needed to avoid mistaking small particles of precipitate for actual colonies of bacteria. To make certain of growth of microorganisms in semisolid medium, however, subplanting to a precipitate-free, dialysate medium is required. By following this method minute microorganisms which are obscured, or simulated, by precipitate in the Smith-Noguchi medium, can be identified.

1. Virulent influenza bacilli, when injected into the nose and throat of monkeys (Cebus capucinus and Macacus syrichtus), excite an acute inflammation of the upper respiratory tract, characterized by swelling and hyperemia of the mucous membrane, infiltration of the mucosa and subrnucosa with leucocytes, desquamation of epithelial cells, and the production of a mucopurulent exudate. The accessory sinuses are often implicated in the infection. 2. Experimental Bacillus influenzae infections of the upper respiratory tract are frequently accompanied or followed by bronchiolitis, peribronchial infiltration, and bronchopneumonia with hemorrhage and edema in the early stage, emphysema and bronchiectasis in the later stages. In general, the process closely resembles uncomplicated Bacillus influenzae pneumonia in man. 3. The injection of virulent influenza bacilli directly into the trachea of monkeys induces in them an experimental bronchiolitis and hemorrhagic bronchopneumonia, similar in all respects to spontaneous Bacillus influenzae pneumonia. 4. In experimental Bacillus influenzaeinfections of either the upper or lower respiratory tract the influenza bacillus can usually be recovered during .the acute stage by culture, either pure or in association with other bacteria. 5. In experimental Bacillus influenzae infections in monkeys characteristic changes occur in the thymus gland-hyperplasia of the follicles, distention of the lymphatic channels, and infiltration of the parenchyma with leucocytes. This enlargement appears to be merely part of a general hyperplasia of the lymphoid structures in the cervical and thoracic regions.

Twelve normal monkeys inoculated on the mucous membranes of the nose or nose and mouth with a strain of Bacillus influenzae; originally isolated in pure culture from the pleural exudate of a case of empyema following influenzal pneumonia in man and subsequently raised in virulence by animal passage, developed an acute self-limited respiratory disease of from 3 to 5 days duration, characterized by sudden onset with profound prostration, the development of rhinitis and tracheobronchitis, with sneezing, cough, and the outpouring of a scanty mucoid, or mucopurulent exudate, a variable febrile reaction, and either a leucopenia or no significant change in the leucocyte count. This disease was complicated in five instances by purulent sinusitis of one or both antra, in three by bronchopneumonia. Bacillus influenzae was recovered at autopsy from the lesions of the disease either in pure culture or in association with organisms that are normal inhabitants of the upper respiratory tract of monkeys. Of ten normal monkeys injected intratracheally with the same strain of Bacillus influenzae, seven developed bronchopneumonia, two developed tracheobronchitis without pneumonia, and one resisted infection. The general symptoms and duration of the disease were similar to those of the preceding group. There were a severe cough and accelerated respirations. Bacillus influenzae was recovered in pure culture from the lungs, bronchi, or trachea in the animals killed during the active stage of the disease. It disappeared promptly from the respiratory tract with recovery. The significance of the first series of experiments in which monkeys were inoculated in the upper respiratory tract is twofold. First, they establish the fact that Bacillus influenzae can initiate in monkeys an acute infection of the normal mucous membranes of the upper respiratory tract; that is, it can act as a primary incitant of respiratory infection without the assistance of a preceding or concomitant contributing cause. In this respect it differs radically from the pneumococcus and Streptococcus haemolyticus, since experiments previously reported(2, 4) have shown that neither of these organisms possesses the property of initiating an infection of the normal mucous membranes of the upper respiratory tract of monkeys, even though the strains used were incalculably more virulent for monkeys than the strain of Bacillus influenzae used in the foregoing experiments. Secondly, the experiments show that Bacillus influenzae infection of the mucous membranes of the upper respiratory tract may spread by continuity to the paranasal sinuses, setting up an acute sinusitis, that it spreads readily to the lower respiratory tract, producing a tracheobronchitis and permitting the ready invasion of secondary bacteria, and that it may penetrate as far as the terminal bronchioles, alveolar ducts, atria, and alveoli, there setting up a bronchiolitis and true bronchopneumonia. In these respects it likewise differs radically from the pneumococcus and Streptococcus haemolyticus which do not possess these pathogenic properties as previous experiments have shown.(2, 4) The bearing of these facts on the possible etiologic relation of Bacillus influenzae to influenza is important, since they show that Bacillus influenzae possesses certain definite primary pathogenic properties which distinguish it and therefore separate it from the group of recognized secondary organisms in influenzal complications, of which the pneumococcus and the streptococcus are the most frequent. The possible etiologic relation of Bacillus influenzae to influenza is further supported by the character of the respiratory disease that occurred in the monkeys. The sudden onset with profound prostration, the absence of leucocytosis or often a leucopenia, the congestion of the mucous membranes of the respiratory tract, the development on the 2nd or 3rd day of an irritative cough due to an inflammatory tracheitis or tracheobronchitis, the brief self-limited course of the infection, and the irregular febrile reactions are all characteristic of influenza. Many of these symptoms were in striking contrast with the symptoms and course of pneumococcus or streptococcus infections in monkeys in which there were no prostration at onset, invariable leucocytosis, and infrequent cough developing only late in the disease. While all the above features of the disease produced in monkeys are characteristic of influenza in man, none are pathognomonic and, in fact, it is doubtful whether uncomplicated influenza possesses any pathognomonic features by which it may be diagnosed certainly in the absence of an epidemic. Even during epidemic times many respiratory infections arise which, though presumably influenza, it is impossible to diagnose as such with certainty. Nor does pathology help in this respect, since there would appear to be no established distinctive lesions of uncomplicated influenza in man, nor for that matter of the complications of influenza, apart from the complications which have been ascribed by Pfeiffer,(5) MacCallum,(6) Wolbach,(7) and others to infection with Bacillus influenzae because of the association of Bacillus influenzae in pure culture with these complications. For these reasons, although the disease produced in monkeys appears to be essentially identical with influenza in man with respect to its clinical course and complications, it is impossible to determine certainly whether it is actually so. The experiments are advanced, therefore, as evidence in favor of the etiologic relation of Bacillus influenzae to influenza, though they do not permit of a definite conclusion in this respect. Their bearing upon the relation of Bacillus influenzae to certain of the complications of influenza would appear to be reasonably conclusive. The recovery of Bacillus influenzae in pure culture at autopsy from the antra, from the trachea and bronchi, and from the lungs in some of the animals developing sinusitis, bronchiolitis, and a characteristic type of bronchopneumonia confirms by animal experiment the etiologic relation of Bacillus influenzae to these complications of influenza, which hitherto has rested solely upon the frequent association of the influenza bacillus with these lesions in man. The production of tracheobronchitis and the same type of bronchopneumonia by the intratracheal injection of Bacillus influenzae in the second series of experiments serves as additional confirmation of this, but has no direct bearing on the etiologic relation of Bacillus influenzae to uncomplicated influenzae.

1. Pneumonia has been consistently produced in normal monkeys by intratracheal injection of Streptococcus haemolyticus. 2. The pneumonia produced has been shown to be comparable with hemolytic streptococcus pneumonia in man with respect to its clinical features, complications, and pathology. 3. Two pathologic types of the disease have occurred, interstitial pneumonia and confluent lobular pneumonia. Both types have been found in the same animal. 4. The type of pneumonia has appeared to be dependent upon the amount of streptococcus culture injected, interstitial pneumonia following the injection of small amounts and being an expression of considerable resistance, confluent lobular pneumonia following the injection of large amounts and being an expression of comparative lack of resistance. 5. Study of the distribution of streptococci in the lungs and of the character of the lesions in early stages of the disease has shown that streptococci may primarily invade the pulmonary tissue by penetration of the walls of the larger bronchial branches and that they are distributed from the points of invasion by way of the peribronchial, perivascular, and septal interstitial tissue and lymphatics. Infection of the alveoli is likewise primarily an interstitial invasion of the alveolar walls by streptococci. 6. In one experiment it was found that preliminary injury to the respiratory tract by gassing with chlorine and that lowering of resistance by a preceding intraperitoneal injection of Bacillus influenza without local injury to the respiratory tract greatly facilitated invasion of the lungs by Streptococcus haemolyticus. 7. A normal monkey inoculated in the nose and throat with Streptococcus haemolyticus failed to develop pneumonia and showed no evidence of infection of the upper respiratory tract.

1. Lobar pneumonia has been consistently produced in normal monkeys by the intratracheal injection of minute amounts of pneumococcus culture. 2. The disease produced has been shown to be clinically identical with lobar pneumonia in man. 3. Lobar pneumonia has been produced in the monkey in one instance by experimental contact infection. 4. Normal monkeys inoculated in the nose and throat with large amounts of pneumococcus culture have failed to develop lobar pneumonia though carrying the organism in their mouths for at least a month. They have likewise failed to show any evidence of upper respiratory tract infection. 5. Monkeys inoculated subcutaneously or intravenously with pneumococcus culture have in no instance developed pneumonia, but have either died of pneumococcus septicemia or recovered without localization of the infection in the lungs.