Discovery of a novel spotted fever group Rickettsia, "Candidatus Rickettsia kedanie," in unfed larval chigger mites, Leptotrombidium scutellare.

Spotted fever group (SFG) rickettsia, the causative agent of SFG rickettsiosis, is predominantly carried by ticks, whereas Orientia tsutusgamushi, the causative agent of scrub typhus, is primarily transmitted by chigger mites in Japan. In this study, we attempted to isolate intracellular eubacteria from Leptotrombidium scutellare, a major vector of O. tsutsugamushi; moreover, we isolated an SFG rickettsia using a mosquito-derived cell line. Draft genome sequences of this unique isolate, by applying criteria for species delimitation, classified this isolate as a novel strain, proposed as "Rickettsia kedanie." Further genetic analysis identified conserved virulence factors, and the isolate successfully propagated in mammalian cells, suggesting its ability to cause diseases in humans. The presence of SFG rickettsia in unfed larvae implies potential dual-pathogen carriage and reflects a symbiotic relationship similar to that between the mites and O. tsutsugamushi, indicating possibility of its transovarial transmission from female adults. Furthermore, conserved genomic similarity of the novel isolate to known SFG rickettsia suggests potential multiple hosts, including chiggers and ticks. In the natural environment, ticks, chigger mites, and wild animals may carry new isolates, complicating the infection cycle and increasing the transmission risks to humans. This discovery challenges the conventional association of SFG rickettsia with ticks, emphasizing its implications for research and disease control. However, this study was confined to a particular species of chigger mites and geographic area, underscoring the necessity for additional studies to comprehend the ecological dynamics, host interactions, and health implications linked to this newly identified SFG rickettsia.

Regulatory Issues: PMDA - Review of Sakigake Designation Products: Oncolytic Virus Therapy with Delytact Injection (Teserpaturev) for Malignant Glioma.

In June 2021, the Ministry of Health, Labor and Welfare approved Delytact Injection as a regenerative medical product for oncolytic virus therapy. The active substance of Delytact Injection is teserpaturev, a genetically engineered herpes simplex virus type 1 (strain F) in which the α47 gene and both copies of the γ34.5 gene have been deleted and the infected cell protein 6 (ICP6) gene has been inactivated by the insertion of the lacZ gene from Escherichia coli. Delytact Injection, when intratumorally administered to patients with malignant glioma, is expected to exert the following effects: (1) the mutant virus selectively replicates in tumor cells and destroys the infected cells through the replication process, exerting a cytocidal effect, and (2) the administration leads to induction of tumor-responsive T cells, which activates antitumor immunity and thus prolongs the survival of patients with malignant glioma. A Japanese phase II study (Study GD01) was conducted in patients with glioblastoma who had residual or recurrent tumors after radiotherapy with concomitant temozolomide. In Study GD01, however, stable disease continued for an extended period in some patients with glioblastoma. Hence, Delytact Injection is expected to be effective to a certain level. In line with this, Delytact Injection has been approved as an option for the treatment of malignant glioma, with one of the 3 approval conditions including conducting a use-results comparison survey and resubmission of the marketing authorization application within the granted time period of 7 years, under the conditional and time-limited approval scheme described in Article 23-26 of Act on Securing Quality, Efficacy and Safety of Products Including Pharmaceuticals and Medical Devices.

Regulatory Aspects of Cell and Gene Therapy Products: The Japanese Perspective.

Regulations for regenerative medicine for human use, such as cell and gene therapy (CGT), have evolved in accordance with advancements in clinical experience, scientific knowledge, and social acceptance of these technologies. In November 2014, two acts, "The Act on the Safety of Regenerative Medicine" (ASRM) and the "Pharmaceuticals, Medical Devices, and Other Therapeutic Products Act" (PMD Act), came into effect in Japan. The responsibilities of medical institutions in ensuring the safety and transparency of such medical technologies are described under ASRM. The PMD Act provides the option of a new scheme for obtaining conditional and time-limited approval for CGT products. Overall, research and development on CGT products, especially gene therapy products, is progressing. New legislative frameworks have been designed to promote the timely development of new technologies and safe and effective CGT products for Japanese patients.

Obligate intracellular bacteria diversity in unfed Leptotrombidium scutellare larvae highlights novel bacterial endosymbionts of mites.

It is well known that the mite Leptotrombidium scutellare carries the pathogen of scrub typhus, Orientia tsutsugamushi. However, our understanding of other bacterial endosymbionts of mites is limited. This study investigated the diversity of the obligate intracellular bacteria carried by L. scutellare using 16S rRNA gene amplicon analysis with next-generation sequencing. The results showed that the detected bacteria were classified into the genera Rickettsia, Wolbachia, and Rickettsiella and an unknown genus of the order Rickettsiales. For further classification of the detected bacteria, a representative read that was most closely related to the assigned taxonomic classification was subjected to homology search and phylogenic analysis. The results showed that some bacteria of the genus Rickettsia were identical or very close to the human pathogens Rickettsia akari, Rickettsia aeschlimannii, Rickettsia felis, and Rickettsia australis. The genetic distance between the genus Wolbachia bacteria in the present study and in previous reports is highly indicative that the bacteria in the present study can be classified as a new taxon of Wolbachia. This study detected obligate intracellular bacteria from unfed mites; thus, the mites did not acquire bacteria from infected animals or any other infectious sources. Finally, the present study demonstrated that various and novel bacterial endosymbionts of mites, in addition to O. tsutsugamushi, might uniquely evolve with the host mites throughout overlapping generations of the mite life cycle. The roles of the bacteria in mites and their pathogenicity should be further examined in studies based on bacterial isolation.

Visualizing developmentally programmed endoreplication in mammals using ubiquitin oscillators.

The majority of mammalian somatic cells maintain a diploid genome. However, some mammalian cell types undergo multiple rounds of genome replication (endoreplication) as part of normal development and differentiation. For example, trophoblast giant cells (TGCs) in the placenta become polyploid through endoreduplication (bypassed mitosis), and megakaryocytes (MKCs) in the bone marrow become polyploid through endomitosis (abortive mitosis). During the normal mitotic cell cycle, geminin and Cdt1 are involved in 'licensing' of replication origins, which ensures that replication occurs only once in a cell cycle. Their protein accumulation is directly regulated by two E3 ubiquitin ligase activities, APC(Cdh1) and SCF(Skp2), which oscillate reciprocally during the cell cycle. Although proteolysis-mediated, oscillatory accumulation of proteins has been documented in endoreplicating Drosophila cells, it is not known whether the ubiquitin oscillators that control normal cell cycle transitions also function during mammalian endoreplication. In this study, we used transgenic mice expressing Fucci fluorescent cell-cycle probes that report the activity of APC(Cdh1) and SCF(Skp2). By performing long-term, high temporal-resolution Fucci imaging, we were able to visualize reciprocal activation of APC(Cdh1) and SCF(Skp2) in differentiating TGCs and MKCs grown in our custom-designed culture wells. We found that TGCs and MKCs both skip cytokinesis, but in different ways, and that the reciprocal activation of the ubiquitin oscillators in MKCs varies with the polyploidy level. We also obtained three-dimensional reconstructions of highly polyploid TGCs in whole, fixed mouse placentas. Thus, the Fucci technique is able to reveal the spatiotemporal regulation of the endoreplicative cell cycle during differentiation.

Fucci-guided purification of hematopoietic stem cells with high repopulating activity.

Fluorescent ubiquitination-based cell cycle indicator (Fucci) technology utilizing the cell cycle-dependent proteolysis of ubiquitin oscillators enables visualization of cell cycle progression in living cells. The Fucci probe consists of two chimeric fluorescent proteins, FucciS/G2/M and FucciG1, which label the nuclei of cells in S/G2/M phase green and those in G1 phase red, respectively. In this study, we generated Fucci transgenic mice and analyzed transgene expression in hematopoietic cells using flow cytometry. The FucciS/G2/M-#474 and FucciG1-#639 mouse lines exhibited high-level transgene expression in most hematopoietic cell populations. The FucciG1-#610 line expressed the transgene at high levels predominantly in the hematopoietic stem cell (HSC) population. Analysis of the HSC (CD34(-)KSL: CD34(-/low)c-Kit(+)Sca-1(+)lineage marker(-)) population in the transgenic mice expressing both FucciS/G2/M and FucciG1 (#474/#610) confirmed that more than 95% of the cells were in G0/G1 phase, although the FucciG1(red) intensity was heterogeneous. An in vivo competitive repopulation assay revealed that repopulating activity resided largely in the FucciG1(red)(high) fraction of CD34(-)KSL cells. Thus, the CD34(-)KSL HSC population can be further purified on the basis of the Fucci intensity.

Stylostome organization in feeding Leptotrombidium larvae (Acariformes: Trombiculidae).

The stylostome of larvae of the trombiculids Leptotrombidium scutellare (Nagayo et al.), Leptotrombidium fletcheri (Womersley et Heaslip) and Leptotrombidium deliense (Walch) was studied experimentally at different time intervals after larval attachment using the histological method. The stylostome of these species has the same organization and belongs to the epidermal combined with the mixed type, developing more in width than in length. Neither transverse nor conspicuous longitudinal layers are present within the stylostome walls, which stain predominantly in red with Azan, also showing longitudinal portions with blue staining. Larvae tend to attach closely to each other and scabs, consisting of the hyperkeratotic epidermal layers fusing with migrating inflammatory cells, develop around the attachment sites. The dermis shows inflammatory foci with dilated capillaries and inflammatory cells inserting in the connective tissue layer underneath the stylostome. The feeding cavity, which is moderately expressed, may be found either in the epidermis or in the dermis. It contains inflammatory cells and their debris in the liquefied host tissues. The stylostome length depends on the character of the attachment site (the thicker epidermis or scab the longer the stylostome), and does not directly correspond to the stages of larval feeding. Nevertheless, at the 48-h time interval, nearly all attached larvae are found to be fully fed and their midgut cells are filled with nutritional globules.

Insights into the clinical development of regenerative medical products through a comparison of three cell-based products recently approved for limbal stem cell deficiency.

Three regenerative medical products for limbal stem cell deficiency (LSCD), a rare and intractable ocular surface disease, have recently been approved in Japan. To our knowledge, this is the first time multiple stem-cell-based medical products have been approved for the same ocular disease. Development plans and study designs for each product differ, resulting in differences in indications. Since cell-based products have a heterogeneous formulation and often target rare diseases, they require a flexible approach to development. This review article describes the status and prospects of the clinical development of regenerative medical products by summarizing the issues of the three products from the Pharmaceuticals and Medical Devices Agency (PMDA) standpoint. Implementing stem cell-based products is challenging, requiring scientific and flexible review by regulatory authorities. To overcome these issues in the development process, developers and regulatory authorities need to communicate and fully discuss study protocols from the early stage of development.

GENETIC VARIATION OF LEPTOTROMBIDIUM (ACARI: TROMBICULIDAE) MITES CARRYING ORIENTIA TSUTSUGAMUSHI, THE BACTERIAL PATHOGEN CAUSING SCRUB TYPHUS.

Leptotrombidium (Acari: Trombiculidae) mites are carriers of Orientia tsutsugamushi, the bacterial pathogen causing scrub typhus in humans. Classification of Leptotrombidium is vital because limited mite species carry O. tsutsugamushi. Generally, Leptotrombidium at the larval stage (approximately 0.2 mm in size) are used for morphological identification. However, morphological identification is often challenging because it requires considerable skills and taxonomic expertise. In this study, we found that the full-length sequences of the mitochondrial cytochrome c oxidase subunit 1 gene varied among the significant Leptotrombidium. On the basis of these, we modified the canonical deoxyribonucleic acid (DNA) barcoding method for animals by redesigning the primer set to be suitable for Leptotrombidium. Polymerase chain reaction with the redesigned primer set drastically increased the detection sensitivity, especially against Leptotrombidium scutellare (approximately 17% increase), one of the significant mites carrying O. tsutsugamushi. Phylogenetic analysis showed that the samples morphologically classified as L. scutellare and Leptotrombidium pallidum were further split into 3 and 2 distinct subclusters respectively. The mean genetic distance (p-distance) between L. scutellare and L. pallidum was 0.2147, whereas the mean distances within each species were 0.052 and 0.044, respectively. Within L. scutellare, the mean genetic distances between the 3 subclusters were 0.1626-0.1732, whereas the distances within each subcluster were 0.003-0.017. Within L. pallidum, the mean genetic distance between the 2 subclusters was 0.1029, whereas the distances within each subcluster were 0.010-0.013. The DNA barcoding uncovered a broad genetic diversity of Leptotrombidium, especially of L. scutellare and L. pallidum, the notable species carrying O. tsutsugamushi. We conclude that the DNA barcoding using our primers enables precise and detailed classification of Leptotrombidium and implies the existence of a subgenotype in Leptotrombidium that had not been found by morphological identification.

Pharmaceuticals and Medical Device Agency approval summary: Amenamevir for the treatment of herpes zoster.

In July 2017, Japan's Ministry of Health, Labor and Welfare issued a marketing authorization valid throughout Japan for N-(2,6-dimethylphenyl)-N-(2-{[4-(1,2,4-oxadiazol-3-yl)phenyl]amino}-2-oxoethyl)-1,1-dioxothiane-4-carboxamide (amenamevir) for the first time worldwide. The decision was based on the favorable opinion of the Pharmaceuticals and Medical Device Agency (PMDA) recommending a marketing authorization of amenamevir for treatment of herpes zoster (HZ). Amenamevir has a different action mechanism from previously approved synthetic nucleoside compounds for the treatment of HZ including acyclovir, valacyclovir and famciclovir. The usual adult dose is 400 mg amenamevir p.o. once daily for 7 days. The benefit is its ability to cure HZ as well as preventing postherpetic neuralgia. The most common side-effects are increase of urine N-acetyl-ß-D-glucosaminidase and α1-microglobulin levels. However, based on the detailed evaluation of the submitted clinical studies, there seems to be no serious safety concerns about amenamevir regarding the kidney of both renally normal and impaired patients. The objective of this article is to summarize the scientific review of the application. The detailed scientific assessment report and product information, including the summary of product characteristics, are available on the PMDA website (www.pmda.go.jp/PmdaSearch/iyakuSearch/).

RNA virome analysis of questing ticks from Hokuriku District, Japan, and the evolutionary dynamics of tick-borne phleboviruses.

Tick-borne viruses have emerged recently in many parts of the world, and the discoveries of novel tick-borne viruses have been accelerated by the development of high-throughput sequencing technology. In this study, a cost-efficient small benchtop next-generation sequencer, the Illumina MiniSeq, was used for the RNA virome analysis of questing ticks collected from Hokuriku District, Japan, and assessed for their potential utility in a tick-borne virus surveillance system. We detected two phleboviruses [Kabuto Mountain virus (KAMV) and Okutama tick virus (OKTV)], a coltivirus [Tarumizu tick virus (TarTV)], and a novel iflavirus [Hamaphysalis flava iflavirus (HfIFV)] from tick homogenates and/or cell culture supernatants after virus isolation processes. The number of sequence reads from KAMV and TarTV markedly increased when cell culture supernatants were used, indicating a successful isolation of these viruses. In contrast, OKTV and HfIFV were detected only in tick homogenates but not from cell culture supernatants, suggesting a failure to isolate these viruses. Furthermore, we performed genomic and phylogenetic analyzes of these detected viruses. OKTV and some phleboviruses discovered recently by NGS-based methods were probably deficient in the M genome segment, which are herein proposed as M segment-deficient phlebovirus (MdPV). A phylogenetic analysis of phleboviruses, including MdPV, suggested that Uukuniemi and Kaisodi group viruses and kabutoviruses evolved from an ancestral MdPV, which provides insights into the evolutionary dynamics of phleboviruses as emerging pathogens.

Hematopoietic stem cell aging is associated with functional decline and delayed cell cycle progression.

The molecular mechanisms underlying hematopoietic stem cell (HSC) aging remain to be elucidated. In this study, we investigated age-related changes in the functional and phenotypic properties of murine HSCs. Consistent with previous studies, we found that the number and frequency of CD34(-/low)c-Kit(+)Sca-1(+)lineage marker(-) (CD34(-)KSL) cells, a highly enriched HSC population, significantly increased in old mice, though their repopulating ability was reduced. Continuous bromodeoxyuridine labeling revealed a significant delay in the cell cycle progression of CD34(-)KSL cells in old mice. This delay was also observed in young recipients transplanted with whole bone marrow cells from old mice. When cultured in vitro, CD34(-)KSL cells from old mice showed a greater capacity to give rise to primitive CD48(-)KSL cells with reduced HSC activity. Gene expression profiling identified age-related changes in the expression of several cell cycle regulatory genes, including p21/Cdkn1a and p18/Cdkn2c. These results support the notion that HSC aging is largely regulated by an intrinsic genetic program.

Repopulating activity of ex vivo-expanded murine hematopoietic stem cells resides in the CD48-c-Kit+Sca-1+lineage marker- cell population.

A better understanding of the biology of cultured hematopoietic stem cells (HSCs) is required to achieve ex vivo expansion of HSCs. In this study, clonal analysis of the surface phenotype and repopulating activity of ex vivo-expanded murine HSCs was performed. After 7 days of culture with stem cell factor, thrombopoietin, fibroblast growth factor-1, and insulin-like growth factor-2, single CD34-/lowc-Kit+Sca-1+lineage marker- (CD34-KSL) cells gave rise to various numbers of cells. The proportion of KSL cells decreased with increasing number of expanded cells. Transplantation studies revealed that the progeny containing a higher percentage of KSL cells tended to have enhanced repopulating potential. We also found that CD48 was heterogeneously expressed in the KSL cell population after culture. Repopulating activity resided only in the CD48-KSL cell population, which had a relatively long intermitotic interval. Microarray analysis showed surprisingly few differences in gene expression between cultured CD48-KSL cells (cycling HSCs) and CD48+KSL cells (cycling non-HSCs) compared with freshly isolated CD34-KSL cells (quiescent HSCs), suggesting that the maintenance of stem cell activity is controlled by a relatively small number of genes. These findings should lead to a better understanding of ex vivo-expanded HSCs.

Generation of cloned mice by direct nuclear transfer from natural killer T cells.

Cloning mammals by nuclear transfer (NT) remains inefficient. One fundamental question is whether clones have really been derived from differentiated cells rather than from rare stem cells present in donor-cell samples. To date, cells, such as mature lymphocytes, with genetic differentiation markers have been cloned to generate mice only via a two-step NT involving embryonic stem (ES) cell generation and tetraploid complementation [1, 2 and 3]. Here, we show that the genome of a unique T-cell population, natural killer T (NKT) cells, can be fully reprogrammed by a single-step NT. The pups and their placentas possessed the rearranged TCR loci specific for NKT cells. The NKT-cell-cloned embryos had a high developmental potential in vitro: Most (71%) developed to the morula/blastocyst stage, in marked contrast to embryos from peripheral blood T cells (12%; p < 1 x 10(-25)). Furthermore, ES cell lines were efficiently established from these NKT-cell blastocysts. These findings clearly indicate a high level of plasticity in the NKT-cell genome. Thus, differentiation of the genome is not always a barrier to NT cloning for either reproductive or therapeutic purposes, so we can now postulate that at least some mammals cloned to date have indeed been derived from differentiated donor cells.

Identification of genes potentially involved in supporting hematopoietic stem cell activity of stromal cell line MC3T3-G2/PA6.

Although coculture of hematopoietic stem cells (HSCs) with stromal cells is a useful system to study hematopoiesis in the niche, little is known regarding the precise cellular and molecular mechanisms of maintaining HSCs through cell-cell interactions. The murine preadipose stromal cell line MC3T3-G2/PA6 (PA6) has been demonstrated to support HSCs in vitro. In this study, microarray analysis was performed on PA6 cells and HSC-nonsupporting PA6 subclone cells to identify genes responsible for supporting HSC activity. Comparison of gene expression profiles revealed that only 144 genes were down-regulated by more than twofold in PA6 subclone cells. Of these down-regulated genes, we selected 11 candidate genes and evaluated for the maintenance of HSC function by overexpressing these genes in PA6 subclone cells. One unknown gene, 1110007F12Rik (also named as Tmem140), which is predicted to encode an integral membrane protein, demonstrated a partial restoration of the defect in HSC-supporting activity.

Isolation and characterization of Tarumizu tick virus: A new coltivirus from Haemaphysalis flava ticks in Japan.

During the course of tick-borne virus surveillance in Japan, three independent isolates of probably the same virus were obtained from three geographically distant populations of the hard tick Haemaphysalis flava. Genome analyses of the three isolates demonstrated that they were closely related but distinct strains of a novel virus, designated Tarumizu tick virus (TarTV), which has a genome of 12 double-stranded RNA segments. The development of the virus-induced cytopathic effects on BHK cells significantly varied according to virus strains. Ten out of 12 segments of TarTV appeared to encode putative orthologs or functional equivalents of viral proteins of Colorado tick fever virus (CTFV) and Eyach virus, suggesting that TarTV is the third member of the genus Coltivirus in the family Reoviridae. This was supported by the facts that the 5'- and 3'-terminal consensus sequences of coltivirus genomes were found also in TarTV genome, and segment 9 of TarTV had sequence and structural features that may mediate a stop codon read-through as observed in that of CTFV. However, segment 7 and 10 of TarTV had no significant sequence similarities to any other proteins of known coltiviruses. Electron microscopic analysis demonstrated that TarTV particle had a non-enveloped bilayer icosahedral structure, and viral inclusion bodies were formed in infected cells. TarTV could infect and replicate in several mammalian cell lines tested, but show no clinical symptoms in intracerebrally inoculated mice. Taken together, our findings provide new insights into genetic diversity and evolution of the genus Coltivirus.

Intestinal parasitic infections in schoolchildren in a suburban area of Hanoi, Vietnam.

An epidemiological study on intestinal parasitic infections among schoolchildren in a suburban area of Hanoi, Vietnam, was conducted. Of the 217 schoolchildren involved in this study, 166 (76%) were positive for at least one of nine species of parasite (six helminths and three protozoa). Among the helminth parasites, Trichuris trichiura (67%) was detected the most frequently followed by Ascaris lumbricoides (34%) and hookworm (3%). In the case of protozoan parasites, Entamoeba coli (8%) was the most frequently detected followed by E. histolytica (2%). No Cryptosporidium parvum or Cyclospora sp were found. A questionnaire survey revealed that there was no positive relationship between parasite infection and the children's school records, educational background or parental income, which have been known to play a role.

DIFFERENTIATION OF PARTIALLY MESODERMALIZED ECTODERM; HOMOIOGENETIC AND HETEROGENETIC INDUCTION BY PRIMARILY INDUCED PART OF THE ECTODERM.

Using embryos of the Japanese newt, Cynops pyrrhogaster, homoiogenetic and heterogenetic induction were investigated in the partially mesodermaelzed presumptive ectoderm. Half of the isolated presumptive ectoderm was placed in contact with the swimbladder of the crucian carp, Carasius auratus, for 15 or 60 min, while the other half was stained with Nile blue sulfate at the same time. The distribution of the stained cells in the tissues evoked in the explants was examined after cultivation for 10 days. Some mesodermal tissues were composed of both stained and unstained cells. This indicates homoiogenetic induction by the primarily induced part of the ectoderm on the other half. The neural and epidermal tissues in the explants were composed of stained cells only, except in one case. We conclude that the neural tissues are derived from cells not placed in contact with the swimbladder and that they are induced by the primarily induced part of the ectoderm.

STAGING OF NEWT BLASTULA EMBRYOS AND FIRST APPEARANCE OF PRIMARY MESODERMAL COMPETENCE.

Using the swimbladder of the crusian carp (Carrasius auratus) as an inductor, the first appearance of mesodermal competence in the presumptive ectoderm of the newt (Triturus pyrrhogaster) blastula was investigated. The time course of embryonic development before the gastrula stage was determined by counting the number of surface cells on a 0.25 mm line at the animal pole. Pregastrula embryos with 2-3, 4-5, 6-7 and 7-8 cells roughly correspond to those at 14, 14-12, 8-6 and 4-0 hr before the beginning of gastrulation. Using presumptive ectoderm of the early gastrula stage, 15 min was found to be the minimum time of contact necessary for the realization of induction. The reactivity of the presumptive ectoderm from pregastrula embryos was tested by 30 min contact. Presumptive ectoderm up to the 4-5 cell stage did not react to the inductor. It may become competent within the next 4-8 hr, since the ectoderm from embryos in the 6-7 cell stage was reactive.

INDUCTIVE CAPACITIES OF GLOMERULAR BASEMENT MEMBRANES AND DENTINE MATRIX.

The inductive capacities of the basement membranes of calf kidney glomeruli and the dentine matrix of the incisors of 23-day rabbit fetuses were examined on the presumptive ectoderm of Triturus gastrulae. The basement membranes caused almost entirely neural induction and the dentine matrix caused mesodermal induction. These findings suggest that intercellular substances play an important role in the inductive effects of heterologous tissues.