Human type I hair keratin pseudogene phihHaA has functional orthologs in the chimpanzee and gorilla: evidence for recent inactivation of the human gene after the Pan-Homo divergence.

In addition to nine functional genes, the human type I hair keratin gene cluster contains a pseudogene, phihHaA (KRTHAP1), which is thought to have been inactivated by a single base-pair substitution that introduced a premature TGA termination codon into exon 4. Large-scale genotyping of human, chimpanzee, and gorilla DNAs revealed the homozygous presence of the phihHaA nonsense mutation in humans of different ethnic backgrounds, but its absence in the functional orthologous chimpanzee (cHaA) and gorilla (gHaA) genes. Expression analyses of the encoded cHaA and gHaA hair keratins served to highlight dramatic differences between the hair keratin phenotypes of contemporary humans and the great apes. The relative numbers of synonymous and non-synonymous substitutions in the phihHaA and cHaA genes, as inferred by using the gHaA gene as an outgroup, suggest that the human hHaA gene was inactivated only recently, viz., less than 240,000 years ago. This implies that the hair keratin phenotype of hominids prior to this date, and after the Pan-Homo divergence some 5.5 million years ago, could have been identical to that of the great apes. In addition, the homozygous presence of the phihHaA exon 4 nonsense mutation in some of the earliest branching lineages among extant human populations lends strong support to the "single African origin" hypothesis of modern humans.

IFN-gamma-mediated prevention of graft-versus-host disease: pharmacodynamic studies and influence on proliferative capacity of chimeric spleen cells.

Recently we demonstrated that prolonged administration of IFN-gamma prevented the development of GVHD in a MHC-mismatched murine BMT model. Treatment with IFN-gamma allowed the development of mature donor-derived allo-tolerant immunocompetent cells in complete chimeric recipients. Here we present data on the pharmacodynamics of this cytokine-mediated protection against GVHD. Treatment with 50000 U IFN-gamma twice weekly for a period of 5 weeks, starting at the day of BMT, was shown to be the optimal treatment protocol, resulting in complete prevention of GVHD-related mortality. Treatment during 1 week with a three-fold higher weekly dose of IFN-gamma (50000 U six times) did not result in significantly improved survival. The start of IFN-gamma administration was a critical factor since a delay of 3 days from the time of BMT resulted in substantial GVHD-induced mortality. Furthermore, it was shown that IFN-gamma treatment inhibited the spontaneous and Con-A-induced proliferation of T cells at 7-14 days after BMT, which is the critical period for the initiation of acute GVHD. However, long-term survivors after IFN-gamma treatment showed a recovery of immunity in contrast to long-term survivors of saline-injected animals, as tested by Con-A responsiveness. It seems that injection of high dose IFN-gamma suppresses the response of potentially alloreactive donor T cells during what normally is the initiation phase of the GVH reaction (GVHR), resulting in the abrogation of GVHD.

Detection of total- and partial-body irradiation in a monkey model: a comparative study of chromosomal aberration, micronucleus and premature chromosome condensation assays.

PURPOSE: To investigate the efficacy of three cytogenetic methods (dicentrics, micronuclei (MN) and premature chromosome condensation (PCC) analysis) for assessment of the unirradiated fraction and the persistence of damage after total-body (TB) and partial-body (PB) irradiation of rhesus monkeys (Macaca mulatta). MATERIALS AND METHODS: Animals were exposed to X-rays (5 Gy), either TB or PB, with about 6% of marrow cells shielded. Blood samples were collected at different times after exposure, i.e. 1, 3 and 7 days, and cultures were set up for the different cytogenetic endpoints. In addition, blood count analysis was performed before and after irradiation. RESULTS: Blood count analysis was not suitable for discriminating between TB and PB exposure. By using Poisson or overdispersion distribution as the basis, it was not possible to distinguish TB from PB irradiation when dicentric chromosomes and MN were analysed. PCC analysis, in contrast, showed a Poisson distribution after TB exposure and overdispersion after PB exposure. Using the PCC assay, reliable dose estimates could be obtained up to 7 days after irradiation. CONCLUSIONS: For dicentrics and MN, shielding of 6% of bone marrow cells was found to be too small to estimate the unirradiated fraction accurately. The PCC technique was useful for dose assessment and the inhomogeneous exposure of 6% was detected within a short period of time after exposure.

Interactions between gut-associated lymphoid tissue and colonization levels of indigenous, segmented, filamentous bacteria in the small intestine of mice.

Unlike most other indigenous bacteria, segmented filamentous bacteria (SFB) are potent activators of the mucosal immune system. SFB are strongly anchored to the epithelial cells of the small intestine where they have a preference for mucosal lymphoid epithelium. Since SFB are only present in high numbers shortly after weaning, it was investigated whether an SFB-induced immune reaction results in the removal of these bacteria from the small intestine. A correlation was found between age and colonization levels in the small intestines of SFB monoassociated Swiss mice. Five-week-old athymic BALB/c (nu/nu) mice showed lower colonization levels than their heterozygous littermates, but the opposite was found at the age of 12 weeks. However, SFB inoculation of germfree Swiss mice resulted in higher colonization levels in 5-week-old mice when compared with 4-month-old mice. We conclude that SFB colonization levels in the small intestine are likely influenced by the activity of the mucosal immune system. However, an additional age-dependent factor that modulates SFB colonization levels cannot be excluded.

Quantification of antigen-specific immunoglobulin A after oral booster immunization with ovalbumin in mice mono-associated with segmented filamentous bacteria or Clostridium innocuum.

Segmented filamentous bacteria (SFB) are known to stimulate the mucosal immune system. Here, the effect of SFB on oral booster immunization with ovalbumin was investigated. Mice mono-associated with SFB or Clostridium innocuum were sensitized by intraperitoneal administration of 100 micrograms ovalbumin with Freunds complete adjuvant. After 4 weeks, mice received 80 mg ovalbumin orally. A maximum IgA response was found 5 days after this booster immunization. Comparison of mice with SFB and mice with C. innocuum revealed a much higher level of IgA in the gut lumen and more IgA secreting cells in the lamina propria of the SFB-associated mice. However, no differences between both groups of animals were found in specific levels of IgA secreting cells or luminal IgA against ovalbumin. It is concluded that there is no enhancing effect of SFB after booster immunization when mice are primed intraperitoneally with ovalbumin.

Increasing the intestinal resistance of rats to the invasive pathogen Salmonella enteritidis: additive effects of dietary lactulose and calcium.

BACKGROUND AND AIMS: Lactulose fermentation by the intestinal microflora acidifies the gut contents, resulting in an increased resistance to colonisation by acid sensitive pathogens. The extent of fermentation should be controlled to prevent acid induced epithelial cell damage. Considering the buffering capacity of calcium phosphate and its intestinal cytoprotective effects, whether supplemental calcium phosphate adds to the increased resistance to intestinal infections by lactulose fermentations was studied. METHODS: In a strictly controlled experiment, rats were fed a purified low calcium control diet, a low calcium/lactulose diet, or a high calcium/lactulose diet, and subsequently infected orally with Salmonella enteritidis. RESULTS: Lactulose fermentation lowered the pH and increased the lactic acid concentration of the intestinal contents, which significantly reduced excretion of this pathogen in faeces; thus it improved the resistance to colonisation. This agreed with the high sensitivity of S enteritidis to lactic acid (main metabolite of lactulose fermentation) in vitro. Calcium phosphate decreased translocation of S enteritidis to the systemic circulation, an effect independent of lactulose. The unfavourable increased cytotoxicity of faecal water caused by lactulose fermentation was more than counteracted by supplemental calcium phosphate. Moreover, calcium phosphate stimulated lactulose fermentation, as judged by the reduced lactulose excretion in faeces and increased lactic acid, ammonia, and faecal nitrogen excretion. CONCLUSION: Extra calcium phosphate added to a lactulose diet improves the resistance to colonisation and translocation of S enteritidis. This is probably mediated by a calcium induced stimulation of lactulose fermentation by the intestinal microflora and reversion of the lactulose mediated increased luminal cytotoxicity, which reduces damage inflicted on the intestinal mucosa.

Interferon-gamma-mediated prevention of graft-versus-host disease: development of immune competent and allo-tolerant T cells in chimeric mice.

Recently it was shown that delayed graft-versus-host disease (GVHD) in mice can be completely prevented by repeated injections of interferon-gamma (IFN-gamma). The characteristics of this sustained IFN-gamma-induced chimerism were studied in more detail. First, the potency of IFN-gamma as a modulator of GVHD was tested in a fully H-2 mismatched murine bone marrow transplantation (BMT) model. Donor bone marrow cells (BMC; C57BL/Rij; H-2b) were mixed with increasing numbers of donor spleen cells (SC) and transplanted into lethally irradiated recipients (C3H/Law; H-2k). Secondly, BMC and SC of the IFN-gamma-induced chimeras (C3H/Law; H-2b) were tested on their immunological competence and GVHD inducing capacity. Repeated injections of the host with IFN-gamma were able to prevent GVHD even when up to 10(5) SC were added to the graft; adding higher numbers of SC resulted in a rapid increase in the frequency of lethal GVHD. Donor-derived lymphocytes (H-2b) obtained from chimeric animals were immunocompetent as concluded from Con A stimulation in vitro. Chimeric-derived BMC (H-2b) were mixed with up to 10(7) chimeric SC (H-2b) and transplanted into a new group of lethally irradiated C3H/Law (H-2k) recipients. All transplanted animals survived the latter treatment without any macroscopic signs of histological lesions typical of GVHD. We conclude that IFN-gamma treatment allows the development of mature donor-derived immunocompetent T cells, which are allo-tolerant for the recipient.

Problems and priorities for controlling opportunistic pathogens with new antimicrobial strategies; an overview of current literature.

An International Study Group on New Antimicrobial Strategies (ISGNAS) has been formed in response to the recognition that development of microbial resistance to antibiotics is becoming a serious, world-wide problem. The group met in 1993 for the first time to discuss the feasibility of developing rational alternatives to the use of antibiotics and prepared, as a result, a comprehensive overview of normal (physiological) mechanisms involved in the control of potentially pathogenic (oppotunistic) microorganisms. One objective of ISGNAS is to understand the conditions which allow opportunistic microbes present among the symbionts to cause an infection. There is a need for more coherent information concerning the habitat, growth requirements and host and pathogen properties which allow opportunistic pathogens to cause life-threatening infections. In particular, information is urgently being sought to understand the complexity of the interactions between the vast number of microbial species, and the interactions between the microbes and their host. Another goal is to inspire and enable basic and clinical research that will lead to the development of new therapies for regulating colonization, translocation and infection by opportunistic micro-organisms in patients during periods of decreased resistance. With a sufficient amount of knowledge of how healthy individuals keep opportunistic micro-organisms under control, it may become feasible for physicians to maintain host resistance and inter-microbial factors involved in the containment of opportunistic microbes. Therapies aimed at boostering natural resistance mechanisms will be of critical importance to individuals whose resistance has been compromised as a result of another clinical condition.

Interferon-gamma prevents graft-versus-host disease after allogeneic bone marrow transplantation in mice.

Lethally irradiated C3H/Law mice were injected (i.v.) with C57BL/Rij allogeneic bone marrow cells to induce a delayed type graft-vs-host disease (GVHD). Signs of GVHD first became apparent in the third week after transplantation. The disease resulted in a mortality rate of 70% at 80 days. Treatment with IFN-gamma twice weekly, for a period of 6 wk, starting at the time of bone marrow transplantation (BMT), completely prevented overt GVHD, as evidenced by a lack of diarrhea and no mortality during the follow-up period of 100 days after BMT. Also, the histologic GVHD lesions in the gastrointestinal tract were almost completely abrogated by the IFN-gamma treatment. All long term survivors were proven to be chimeras. During the induction phase of GVHD, the number of Con A-induced, IFN-gamma-producing cells in the spleen was significantly reduced in the IFN-gamma-treated mice as compared with control mice. These results suggest that the normally enhanced production of endogenous IFN-gamma in the spleen at the time of hematopoietic reconstitution after BMT is down-regulated by exogenously administered IFN-gamma. This cytokine-mediated strategy to prevent GVHD might be an alternative to the current strategy of in vitro depletion of T cells for allogeneic BMT.

Long-term expression of human adenosine deaminase in rhesus monkeys transplanted with retrovirus-infected bone-marrow cells.

Gene transfer into hemopoietic stem cells could offer a lasting cure for a variety of congenital disorders. As a preclinical test for such a gene therapy, rhesus monkeys were transplanted with autologous bone-marrow cells infected with helper-free recombinant retroviruses carrying the human adenosine deaminase gene. The in vivo regenerative capacity of the infected bone marrow could be conserved, suggesting survival of repopulating hemopoietic stem cells. In the hemopoietic system of transplanted animals the foreign gene could be observed for as long as the animals were analyzed (in two monkeys greater than 1 yr after transplantation). Genetically modified cell types and tissues included peripheral blood mononuclear cells, granulocytes, bone-marrow cells of various densities, and spleen and lymph nodes. The presence of the provirus in the short-living granulocytes greater than 1 yr after bone-marrow transplantation provided evidence for the transduction of very primitive hemopoietic progenitors. Moreover, the gene transfer resulted in sustained production of functional human adenosine deaminase enzyme in peripheral blood mononuclear cells. These results demonstrate the feasibility of bone-marrow gene-therapy approaches, in particular for treating adenosine deaminase deficiency.

Experimental and clinical gnotobiotics: influence of the microflora on graft-versus-host disease after allogeneic bone marrow transplantation.

One of the major complications of allogeneic bone marrow transplantation (BMT) is graft-versus-host disease (GvHD), which is caused by donor type lymphocytes which react against the recipient's tissues. An important factor which influences GvHD is the recipient's gastrointestinal microflora. This was originally observed in gnotobiotic mice. Infusion of 10(7) H-2 incompatible bone marrow cells into lethally irradiated (9.0 Gy X-rays) conventional mice results in a late onset type GvHD which causes the death of the majority of the recipients during the first two months after BMT. This mortality can be completely prevented if the recipients are germfree mice, or when they are conventional animals which have been subjected to complete or selective gastrointestinal decontamination (GID). In a mouse model, the mechanism responsible for the influence of the microflora on GvHD after allogeneic BMT was investigated. These studies indicate that GvHD can be induced by activated T-lymphocytes from donor origin reacting against bacterial antigens which might be cross-reactive with the recipient's epithelial tissue antigens. Activation of these T-lymphocytes is confined to antigens of certain bacterial species of the recipient which are not present in the indigenous microflora of the donor mice. These bacteria most likely belong to the anaerobic flora of the recipient. The latter hypothesis is strongly supported by the observation in human patients that, in contrast to complete GID, selective decontamination of the gastrointestinal tract did not have any beneficial effect on moderately severe to severe GvHD after transplantation with MHC-matched sibling donor bone marrow grafts.

The use of a rat-derived microflora for providing colonization resistance in SPF rats.

To obtain a suitable species-specific microflora for a new rat SPF-unit, germ-free WAG/Rij rats were associated with a flora derived originally from selectively decontaminated Cpb: WU (Wistar) rats. Caecal and ileal contents of these rats had been cultured anaerobically (37 degrees C) for 7 days and harvested. This cultured flora was given to germ-free Cpb: SE (Swiss) mice, which were kept in an isolator system and acted as a source of the flora to associate germ-free Wag/Rij rats. In these associated rats, several parameters indicative of the 'quality' of the intestinal microflora were investigated and compared to those in rats with a mouse derived anaerobic microflora. Parameters included relative caecal weight, colonization resistance and the concentration of faecal bile acids. The cultured rat-derived microflora normalized the observed intestinal parameters better than the mouse derived microflora, and provided better colonization resistance. We conclude that culturing of intestinal contents of selectively decontaminated animals can be a useful way to obtain a species-specific donor-microflora which can be used to start new SPF units.

Prevention of infection and graft-versus-host disease by suppression of intestinal microflora in children treated with allogeneic bone marrow transplantation.

The effect of suppression with antimicrobial agents of the intestinal microflora of paediatric bone marrow graft recipients on severe bacterial and fungal infections and on moderate to severe acute graft-versus-host disease was studied retrospectively. Data on 65 cases of bone marrow transplantation for either severe bone marrow failure or leukaemia, performed in a strict protective environment with either complete or selective gastrointestinal decontamination, were evaluated. All bone marrow grafts were from HLA-identical siblings and were not depleted of T-lymphocytes. Twenty percent of the recipients had one or more episodes of septicaemia during the granulocytopenic period after transplantation, mostly due to gram-positive bacteria. Only five children died due to infection, in each case caused by a microorganism originating from the endogenous flora. Complete gastrointestinal decontamination was superior to selective gastrointestinal decontamination in preventing infectious complications (p less than 0.001). The same was the case for the prevention of acute graft-versus-host disease of grade II or higher, which was observed in 7 of 40 (17.5%) completely decontaminated children versus 9 of 18 (50%) selectively decontaminated children evaluable for graft-versus-host disease (p less than 0.01). It is concluded that complete gastrointestinal decontamination in a strict protective environment is a feasible and very effective method for preventing severe infections and acute graft-versus-host disease after allogeneic bone marrow transplantation in children and adolescents; it resulted in a low transplantation-related mortality of 26% and a good quality of survival in 69% of the graft recipients.

Background (spontaneous) immunoglobulin production in the murine small intestine before and after weaning.

The ontogeny of the murine intestinal B-cell compartment before and after weaning was studied by quantitative analysis of immunoglobulin-secreting cells (Ig-SC) in the small intestine (SI). Before weaning, few Ig-SC were detected in the SI, whereas spleen and bone marrow already contained many Ig-SC. The number of Ig-SC in the SI started to increase immediately after weaning. Comparing early-weaned mice with non-weaned mice of the same age clearly demonstrated that weaning brought on the development of Ig-SC in the SI. The influence of a gut flora on the number of Ig-SC in the SI was examined by comparing the number of Ig-SC in the SI of conventionally housed, specific pathogen free (SPF) and germ-free mice. A bacterial flora was apparently needed for the normal development of Ig-SC in the SI. Comparing mice containing an aerobic Gram-negative bacterial flora with mice containing only an anaerobic Gram-positive bacterial flora demonstrated that the type of bacterial flora is relatively unimportant. No evidence was found that circulating maternal antibodies suppressed the development of the "spontaneous" intestinal and systemic B cell response. The results show that bacterial colonization of the intestine plays a pivotal role in the development of the Ig-SC compartment in the SI.

Management of bacterial and fungal infections in bone marrow transplant recipients and other granulocytopenic patients.

A review of studies on the effect of different types of gastrointestinal decontamination and protective environment on infectious complications in granulocytopenic patients is given, and the effect of these measures on graft-versus-host disease after allogeneic bone marrow transplantation is discussed. It is concluded that complete gastrointestinal decontamination of patients nursed under conditions of strict reverse isolation will maximally prevent infections, graft-versus-host disease, and lung complications and therefore is the treatment to be preferred for patients undergoing bone marrow transplantation. Since selective decontamination is as effective in preventing bacterial and fungal infections as is complete decontamination, this treatment is to be preferred for other patients with a greatly reduced resistance to these infections. The reason is that, for this type of patient, selective decontamination can be performed without the use of strict isolation facilities and in this way is less laborious and less of a burden for the patient. Besides this, the number of patients that can be treated will not be limited by the number of available facilities for strict reverse isolation, which can be reserved for bone marrow transplant patients.

Idiopathic paraproteinaemia. IV. The role of genetic factors in the development of monoclonal B cell proliferative disorders--a study in the ageing C57BL/KaLwRij and CBA/BrARij mouse radiation chimeras.

Mouse radiation chimeras, employing strains with a low (CBA/BrARij) and a high (C57BL/KaLwRij) frequency of idiopathic paraproteinaemia (IP), were used in a study on genetic influences in the development of IP, a benign B cell monoclonal proliferative disorder. Taking advantage of the different Igh1 allotypic markers between the two strains, the development of IP with increasing age was investigated by agar electrophoresis, immunoelectrophoresis and immunofixation. Four of 18 CBA recipients transplanted with C57BL bone marrow cells were shown to develop IP of the IgG2a isotype and the Igh1b (donor) allotype during their life. In contrast, none of the 23 C57BL recipients of CBA bone marrow developed an IgG2a paraprotein of the Igh1a allotype. However, in three of these 23 chimeras, an IgG2a and Igh1b (recipient) allotype paraprotein appeared with age; two of these mice proved to be reversals at 12 months and one at 15 months of age. The frequencies of homogeneous immunoglobulins of the donor type in the chimeras corresponded roughly to those of normal mice of the donor strain. Histopathological examination excluded a malignant origin of these monoclonal proliferations. These findings support the view that intrinsic cellular genetic factors are of major importance in the development of IP, a benign B cell neoplasia.

Clinicopathologic study of six cases of meningitis and meningoencephalitis in chimpanzees (Pan troglodytes).

Three fatal cases of purulent meningitis and one fatal case of thromboembolic necrotizing meningoencephalitis occurred in chimpanzees from the Primate Center TNO, The Netherlands. In addition, two apes had clinical signs of meningitis and were successfully treated. The severity of the residual hemiparesis and dysphagia in one of these two apes was such that it was killed for humane reasons. The histopathological diagnosis was chronic active meningoencephalitis. Streptococcus pneumoniae was isolated from five apes and Klebsiella pneumoniae from one. In the majority of cases, the primary site of infection was the upper respiratory tract. After reducing the population density, initiating a vaccination program using a commercially available human polyvalent pneumococcal vaccine, and changing the cleaning procedure of the animal facilities, no other cases of meningitis or meningoencephalitis have occurred in the chimpanzee colony in the ensuing 3.5 years.

Applicability of solidified water (hydrogel) in laboratory animal care.

Hydrogel was used to provide water in a solid state to rats and mice under laboratory conditions. Hydrogel was easy to handle and readily consumed by the animals. In studies extending over 10 months, no adverse effects on the condition of the animals were observed. Consumption of hydrogel as the sole source of water for nearly 2 months had no effect on the concentration of selected fecal aerobic microorganisms. Histopathological changes were not observed in the tissues of rats and mice kept for periods up to 7 months on hydrogel as the sole source of fluids. The survival time of rats and mice kept on hydrogel after exposure to supralethal doses of total body irradiation did not differ significantly from that of control animals given drinking water in bottles.