Raman spectroscopy predicts the link between claw keratin and bone collagen structure in a rodent model of oestrogen deficiency

Osteoporosis is a common disease characterised by reduced bone mass and an increased risk of fragility fractures. Low bone mineral density is known to significantly increase the risk of osteoporotic fractures, however, the majority of non-traumatic fractures occur in individuals with a bone mineral density too high to be classified as osteoporotic. Therefore, there is an urgent need to investigate aspects of bone health, other than bone mass, that can predict the risk of fracture. Here, we successfully predicted association between bone collagen and nail keratin in relation to bone loss due to oestrogen deficiency using Raman spectroscopy. Raman signal signature successfully discriminated between ovariectomised rats and their sham controls with a high degree of accuracy for the bone (sensitivity 89%, specificity 91%) and claw tissue (sensitivity 89%, specificity 82%). When tested in an independent set of claw samples the classifier gave 92% sensitivity and 85% specificity. Comparison of the spectral changes occurring in the bone tissue with the changes occurring in the keratin showed a number of common features that could be attributed to common changes in the structure of bone collagen and claw keratin. This study established that systemic oestrogen deficiency mediates parallel structural changes in both the claw (primarily keratin) and bone proteins (primarily collagen). This strengthens the hypothesis that nail keratin can act as a surrogate marker of bone protein status where systemic processes induce changes.     

Immunocytochemical localization and biochemical analysis of alpha and beta keratins in the avian lingual epithelium

The alpha and beta keratins are found as 10-nm and 3-nm cytoplasmic filaments, respectively. While the alpha keratins are produced in essentially all vertebrate epithelia (Franke et al.: Exp. Cell Res., 116:429-445, 1978; Sun et al.: Proc. Natl. Acad. Sci. USA, 76:2813-2817, 1979), the beta keratins have been demonstrated only in specific epithelial tissues of birds and reptiles (Sawyer et al.: In: Biology of the Integument: Vertebrates. J. Bereiter-Hahn, A.G. Matoltsy, and K.S. Richards, eds. Springer-Verlag, Berlin, Vol. 2, pp. 194-238, 1986; Landmann: In: Biology of the Integument: Vertebrates. J. Bereiter-Hahn, A.G. Matoltsy, and K.S. Richards, eds. Springer-Verlag, Berlin, Vol. 2, pp. 150-187, 1986). Recently, Homberger and Brush (Zoomorphology, 106:103-114, 1986) have demonstrated that within the lingual epithelium of parrots, beta keratins are expressed exclusively in the anterior ventral region. While it is well established that epidermal-dermal interactions are important for the regional expression of the beta keratin genes in the avian scutate scales and feathers, little is known about the expression of beta keratins in other epithelial structures such as the tongue. We have used biochemical and immunocytochemical techniques to analyze the alpha and beta keratins of the lingual epithelium of the chick as an initial step in the characterization of this model system for developmental studies. We have found that alpha keratins are present throughout the lingual epithelium. The anterior ventral epithelium contains alpha keratin polypeptides characteristic of skin-type differentiation, while the epithelium of the dorsal and posterior ventral regions contains alpha keratin polypeptides characteristic of esophageal-type differentiation (O'Guin et al.: In: Current Topics in Developmental Biology: The Molecular and Developmental Biology of Keratins. A.A. Moscona and A. Monroy, eds. R.H. Sawyer, vol. ed. Academic Press, New York, Vol. 22, pp. 282-306, 1987). Beta keratins are produced only in the differentiated epithelial cells of the anterior ventral region of the tongue. Immunoelectron microscopy demonstrates that the alpha and beta keratins of the stratum intermedium and corneum of the anterior ventral region are found together in the large filament bundles characteristic of this region. The preexistence of the alpha keratins in the cells destined to produce beta keratins as well as the colocalization of these keratins in the filament bundles of these cells suggests that a functional relationship may exist between the alpha and beta keratins.     

Histological microstructure of the claws of the African clawed frog, Xenopus laevis (Anura: Pipidae): implications for the evolution of claws in tetrapods

Claws are consistent components of amniote anatomy and may thus be implicated in the success of the amniote invasion of land. However, the evolutionary origin of these structures in tetrapods is unclear. Claws are present in certain extant non-amniotes, such as Xenopus laevis, the African clawed frog. The histology of the soft tissue component of the claws of X. laevis is described and compared with the amniote condition in order to gain new information on the question of homology of claws in these two groups based on patterns of keratinization.The X. laevis claw sheath is composed of a localized thickening of the corneous region of the epidermis that envelops the terminal phalanx. Noted differences between the non-cornified layers of the epidermis of the claw and non-claw region are the overall grainier appearance of the cells and an increased abundance of desmosomes in the intermediate spinosus cells. The biochemical identity of the sheath keratin(s) is inferred to be different from that of non-claw region epidermis, based on histological differences and differences in stain affinity between the two regions. The microstructure of the frog claw differs from that of amniotes in several respects, including the lack of a specified zone of growth near the base of the claw. Amphibians and amniotes, therefore, have very different patterns of claw sheath growth. Observations do not support homology of claws on a structural level in these two groups; however, further experimental work may confirm a conserved pattern of cornification in these structures in tetrapods.     

Size,but not sex,predicts pinch force and exoskeleton mechanical properties in crayfish of the genus Faxonius

Studies of animal weaponry and defensive structures rarely take into consideration their underlying mechanical properties. We measured the compressive strength and thickness of the exoskeleton of the claw (chela) in two North American crayfish species, Faxonius virilis and F. limosus. We performed similar measures on the carapace, a body region not directly involved in agonistic contests. Males of both species generated significantly stronger maximum pinch forces than females. However, these differences can be attributed to differences in claw size between the sexes. The thickness (ultrastructure) of the claw exoskeleton was a significant predictor of its compressive strength and likely explained the difference in compressive strength we observed between the two species. Neither claw thickness nor claw compressive strength was correlated with maximum pinch force. Additionally, we found that crayfish body size was a strong predictor of carapace compressive strength and thickness, whereas sex was not. The claw had greater compressive strength and thickness than the corresponding values for the carapace. Our study shows that the mechanical properties of the crayfish exoskeleton are largely a function of size and highlights the need to integrate mechanical properties into studies of animal morphology and performance.     

Ultrastructural localization of hair keratin homologs in the claw of the lizard Anolis carolinensis

The claw of lizards is largely composed of beta‐keratins, also referred to as keratin‐associated beta‐proteins. Recently, we have reported that the genome of the lizard Anolis carolinensis contains alpha keratin genes homologous to hair keratins typical of hairs and claws of mammals. Molecular and immunohistochemical studies demonstrated that two hair keratin homologs named hard acid keratin 1 (HA1) and hard basic keratin 1 (HB1) are expressed in keratinocytes forming the claws of A. carolinensis. Here, we extended the immunocytochemical localization of the novel reptilian keratins to the ultrastructural level. After sectioning, claws were subjected to immunogold labeling using antibodies against HA1, HB1, and, for comparison, beta‐keratins. Electron microscopy showed that the randomly organized network of tonofilaments in basal and suprabasal keratinocytes becomes organized in long and parallel bundles of keratin in precorneous layers, resembling cortical cells of hairs. Entering the cornified part of the claw, the elongated corneous cells fuse and accumulate corneous material. HA1 and HB1 are absent in the basal layer and lower spinosus layers of the claw and are expressed in the upper and precorneous layers, including the elongating corneocytes. The labeling for alpha‐keratin was loosely associated with filament structures forming the fibrous framework of the claws. The ultrastructural distribution pattern of hard alpha‐keratins resembled that of beta‐keratins, which is compatible with the hypothesis of an interaction during claw morphogenesis. The data on the ultrastructural localization of hair keratin homologs facilitate a comparison of lizard claws and mammalian hard epidermal appendages containing hair keratins. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

A microscopic study of the marmoset claw and nail

W. E. Le Gros Clark concluded in his study of the problem of the primate claw that the essential difference between claw and nail is the presence of a terminal matrix associated with a deep layer in the claw, whereas neither terminal matrix nor deep layer exists in the nail. He demonstrated that the marmoset claw (which tips every digit except the hallux) has a thin deep layer and a recognizable terminal matrix. The present paper reports and discusses evidence that the marmoset nail also has a deep layer and terminal matrix. Although the importance of these structures in the claw is not disputed, it appears that these can no longer be considered absolute differences between claw and nail. On the basis of this evidence, it cannot be claimed that the presence of the deep layer and terminal matrix determines the distinctive shape of the claw as opposed to the nail.     

The keratin polypeptide patterns in heterotypically recombined epithelia of skin and mucosa of adult mouse

Epithelial-mesenchymal interactions were investigated considering both morphologic criteria and keratin polypeptide expression in homotypic and heterotypic recombinants of adult mouse skin and oral mucosa. Two series of cross-recombinants of epithelia with different morphology and keratin patterns were chosen: (a) footpad epidermis/ear dermis and ear epidermis/footpad dermis; (b) palate epithelium/cheek connective tissue and cheek epithelium/palate connective tissue. Homotypic and heterotypic recombinants were prepared after EDTA-separation of the original tissues and then grown on syngeneic mice in subcutaneously prepared protected graft chambers. EDTA-separation is especially suited to completely separate the epidermal-dermal union, and the transplantation procedure used strictly prevents contamination with host epithelium. Five weeks after implantation keratins were analyzed by one and two-dimensional gel electrophoresis and peptide mapping. In both series, homotypic recombination of the tissues did not alter the original morphology and keratin polypeptide composition of the individual epithelial components. Ear epidermis displayed no significant changes in structure or keratin pattern in heterotypic recombinants. Recombined with ear dermis, footpad epidermis showed acquisition of some morphologic features typical for ear epidermis and slight changes in keratin composition which were, however, difficult to interpret due to the normal similarities of footpad keratin with that of ear. In contrast, the heterorecombinants of the palate/cheek series exhibited considerable alterations in their keratin patterns. Either epithelium showed suppression of distinct keratin subunits and de novo expression of subunits characteristic of the epithelium normally associated with the connective tissue component. The keratin patterns of both matches closely resembled each other and represented patterns intermediate between the normal patterns. This partial, however, significant modulation in the expression of differentiation markers was paralleled by similarly directed changes in the architecture of the heterotransplanted tissues, thus indicating that both morphogenesis and cytodifferentiation of certain adult epithelia can be influenced by extrinsic mesenchymal factors.     

Keratin protein expression during the development of conducting airway epithelium in nonhuman primates

Keratin protein expression during the development of Rhesus monkey conducting airway epithelium was investigated by both biochemical and immunohistochemical methods. Keratin proteins were extracted from tracheal and intrapulmonary airway tissues of fetal (at 80- and 140-day gestational ages), neonatal, and adult animals. Using immunoblot analyses and immunohistochemistry with various monoclonal (AE1, AE3, AE8, 6.01 and 6.11) and monospecific antibodies (anti-50/55 and anti-40 kDa), the presence of keratins 5, 6, 8, 13, 14, and 19 in adult airway epithelium were demonstrated. Except for keratin 13 (51 kDa), the remaining keratins could be immunologically detected in fetal and neonatal tissues. To further understand the nature of the synthesis of keratin 13 during development, airway epithelial cells from different ages were isolated and cultured in vitro. Cultured cells were labeled with 35S-methionine, and the patterns of keratin protein were analyzed by one- and two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results indicated that the cultured airway cells synthesized additional keratins including 7, 15, 16, 17, and 18. However, consistent with the in vivo finding, fetal cells synthesized less or no keratin 13. These in vivo and in vitro studies strongly suggest that the synthesis of the keratin 13 in monkey conducting airway epithelium is developmentally regulated.     

Region-specific expression of scutate scale type beta keratins in the developing chick beak.

This study shows that different patterns of scutate scale type beta keratins are accumulated in the three adjacent structures of the embryonic chick beak: periderm, egg tooth, and cornified beak. The cornified beak accumulates all of the beta keratins of scutate scale except pp2,3. The periderm, which is the outermost, multilayered covering of the whole embryonic beak, accumulates only beta keratins 2,3, and p2,3 of the scutate scale pattern. The egg tooth, which is the rounded elevation on the dorsal surface of the upper beak, and the embryonic claw accumulate greatly reduced levels of 2,3 and p2,3 compared to scutate scale. Like cornified beak, the claw does not accumulate pp2,3, but both tissues express a potentially new beta keratin, beta keratin 8. Neither the histidine rich "fast" proteins (HRPs), which are expressed in embryonic scutate scales and feathers, nor the avian cytokeratin associated proteins (cap-1 and cap-2), which are expressed in scutate and reticulate scales, are expressed in any of the embryonic beak structures or in the claw. The implications of these findings with regard to regulation of terminal differentiation of avian skin are discussed.     

Keratin biosynthesis in normal mouse epithelia and in squamous cell carcinomas. mRNA-dependent alterations of the primary structure of distinct keratin subunits in tumors

The keratin polypeptide patterns of two murine transplantable squamous cell carcinomas--originally induced by chemical means in the back skin and in the forestomach epithelium--are deficient in high molecular weight keratin subunits (greater than 60 kDa) invariably present in the corresponding normal tissues. In addition, the keratin polypeptide composition within the low molecular weight range showed further alterations with regard to the corresponding keratin subset of normal tissues in that both tumors expressed a 40-kDa protein, and a 56-kDa protein was selectively found in the forestomach tumor. A comparison of the charge properties of normal and tumor keratin polypeptides revealed that the two uppermost tumor proteins at 60 and 58 kDa were basic in nature whereas their normal molecular weight counterparts belonged to the acidic subset of the pattern. These tumor proteins also showed mutually identical peptide maps which, however, were considerably different from those of the normal proteins. The remaining tumor keratin subunits at 52, 50, 48, and 45 kDa, common also to the normal tissues, had retained their normal charge properties. In vitro translation of mRNA, isolated from both normal and tumor tissue, revealed that every tumor keratin polypeptide is encoded by its own mRNA. In contrast to normal keratinizing tissues, there is therefore no indication of post-translational protein processing in tumors. The in vitro translation products of tumor RNAs had all properties in common with the in vivo tumor proteins, thus indicating that every deviation of the tumor keratin spectrum from the normal state is determined at the mRNA level.     

Microanatomy of passerine hard‐cornified tissues: Beak and claw structure of the black‐capped chickadee (Poecile atricapillus)

The microanatomy of healthy beaks and claws in passerine birds has not been well described in the literature, despite the importance of these structures in avian life. Histological processing of hard‐cornified tissues is notoriously challenging and only a few reports on effective techniques have been published. An emerging epizootic of beak deformities among wild birds in Alaska and the Pacific Northwest region of North America recently highlighted the need for additional baseline information about avian hard‐cornified structures. In this study, we examine the beak and claw of the Black‐capped Chickadee (Poecile atricapillus), a common North American passerine that is affected by what has been described as “avian keratin disorder.” We use light and scanning electron microscopy and high‐magnification radiography to document the healthy microanatomy of these tissues and identify features of functional importance. We also describe detailed methods for histological processing of avian hard‐cornified structures and discuss the utility of special stains. Results from this study will assist in future research on the functional anatomy and pathology of hard‐cornified structures and will provide a necessary reference for ongoing investigations of avian keratin disorder in Black‐capped Chickadees and other wild passerine species. © 2011 Wiley Periodicals, Inc.     

Desmoplakin controls microvilli length but not cell adhesion or keratin organization in the intestinal epithelium

Maintaining proper cell-cell adhesion in the intestine is essential for tissue homeostasis and barrier function. This adhesion is thought to be mediated by cell adhesion structures, including tight junctions, adherens junctions, and desmosomes, which concentrate in the apical junctional region. While clear roles for adherens and tight junctions have been established in simple epithelia, the function of desmosomes has not been addressed. In stratified epithelia, desmosomes impart mechanical strength to tissues by organizing and anchoring the keratin filament network. In this paper, we report that the desmosomal protein desmoplakin (DP) is not essential for cell adhesion in the intestinal epithelium. Surprisingly, when DP is lacking, keratin filament localization is also unperturbed, although keratin filaments no longer anchor at desmosomes. Unexpectedly, DP is important for proper microvillus structure. Our study highlights the tissue-specific functions of desmosomes and reveals that the canonical functions for these structures are not conserved in simple epithelium.     

Glycoconjugates and keratin 18 define subsets of taste cells

Summary Sections of neonatal, normal adult and denervated adult rat tongue were examined with lectin histochemistry. Attention was focused upon intragemmal cells (cells within the taste bud) and the surrounding perigemmal cells. Informative staining patterns were observed with four of 12 lectins:Ulex europaeus (UEA-I),Bauhinia purpurea (BPA),Helix pomatia (HPA) andLotus tetragonolobus (LTA) agglutinins. In normal adult tongues, BPA bound to those lingual epithelial cells lacking contact with the basal lamina. After they formed, vallate taste buds were laterally surrounded by distinctive BPA-positive cells. HPA reacted selectively with 28% and LTA with 23% of the intragemmal cells in vallate/foliate taste buds. In double-stained taste buds there was, a statistically significant overlap of LTA-positive cells and keratin 18-positive cells. The overlap between HPA binding and keratin 18 was more marked: double-stained cells comprized 67% of all stained cells. During taste bud development in neonates keratin 18 synthesis preceded HPA binding. In contrast, during the replacement of adult taste cells, keratin 18 synthesis and HPA binding were generally concurrent. Keratin 18 and HPA probably identify the same subset of older taste receptor cells. HPA may bind to glycoconjugates on the surface of keratin 18-positive cells. In denervated adult tongue the loss of all UEA-I-positive or BPA-positive perigemmal cells suggests that perigemmal as well as intragemmal cells are nerve-dependent.     

Evolution and development of mammalian limb integumentary structures

The adaptive radiation of mammalian clades has involved marked changes in limb morphology that have affected not only the skeleton but also the integumentary structures. For example, didelphid marsupials show distinct differences in nail and claw morphology that are functionally related to the evolution of arboreal, terrestrial, and aquatic foraging behaviors. Vespertilionoid bats have evolved different volar pad structures such as adhesive discs, scales, and skin folds, whereas didelphid marsupials have apical pads covered either with scales, ridges, or small cones. Comparative analysis of pad and claw development reveals subtle differences in mesenchymal and ectodermal patterning underlying interspecific variation in morphology. Analysis of gene expression during pad and claw development reveals that signaling molecules such as Msx1 and Hoxc13 play important roles in the morphogenesis of these integumentary structures. These findings suggest that evolutionary change in the expression of these molecules, and in the response of mesenchymal and ectodermal cells to these signaling factors, may underlie interspecific differences in nail, claw, and volar pad morphology. Evidence from comparative morphology, development, and functional genomics therefore sheds new light on both the patterns and mechanisms of evolutionary change in mammalian limb integumentary structures.     

Effects of subepithelial fibroblasts on epithelial differentiation in human skin and oral mucosa: heterotypically recombined organotypic culture model

The stratified squamous epithelia differ regionally in their patterns of morphogenesis and differentiation. Although some reports suggested that the adult epithelial phenotype is an intrinsic property of the epithelium, there is increasing evidence that subepithelial connective tissue can modify the phenotypic expression of the epithelium. The aim of this study was to elucidate whether the differentiation of cutaneous and oral epithelia is influenced by underlying mesenchymal tissues. Three normal skin samples and three normal buccal mucosa samples were used for the experiments. Skin equivalents were constructed in four ways, depending on the combinations of keratinocytes (cutaneous or mucosal keratinocytes) and fibroblasts (dermal or mucosal fibroblasts), and the effects of subepithelial fibroblasts on the differentiation of oral and cutaneous keratinocytes were studied with histological examinations and immunohistochemical analyses with anti-cytokeratin (keratins 10 and 13) antibodies. For each experiment, three paired skin equivalents were constructed by using single parent keratinocyte and fibroblast sources for each group; consequently, nine (3 x 3) organotypic cultures per group were constructed and studied. The oral and cutaneous epithelial cells maintained their intrinsic keratin expression. The keratin expression patterns in oral and cutaneous epithelia of skin equivalents were generally similar to their original patterns but were partly modified exogenously by the topologically different fibroblasts. The mucosal keratinocytes were more differentiated and expressed keratin 10 when cocultured with dermal fibroblasts, and the expression patterns of keratin 13 in cutaneous keratinocytes cocultured with mucosal fibroblasts were different from those in keratinocytes cocultured with cutaneous fibroblasts. The results suggested that the epithelial phenotype and keratin expression could be extrinsically modified by mesenchymal fibroblasts. In epithelial differentiation, however, the intrinsic control by epithelial cells may still be stronger than extrinsic regulation by mesenchymal fibroblasts.     

Local variations in the staining patterns of keratin antigens and blood group antigens in normal rodent oral epithelia

Summary All rodent oral epithelia are orthokeratinized. However, morphological, immunohistochemical and biochemical studies have shown that regional differences exist. In the present study, intraregional variations in differentiation patterns of rat oral epithelia are demonstrated using monoclonal anti-keratin antibodies AE1 and AE2 and antibodies to blood group antigens B and H. Well-defined areas of rat buccal and hard palate epithelium differed from the general staining patterns of these epithelia. These areas were associated with a papillary surface contour. These local variations were not found in the strain of mice examined. The results suggest that physiologically different vertical compartments of keratinocytes exist within one and the same region of rat oral mucosa, a phenomenon previously recognized in detail only in the epithelium of dorsal tongue. The papillary structures may have some functional significance related to the processing of food similar to that suggested for lingual filiform papillae.     

Morphogenesis of rat lingual filiform papillae.

The morphogenesis of filiform papillae on rat tongue was investigated with the electron microscope. Tongue rudiments were first seen on the 12th day of gestation. At 15-17 days, dermal papillae had formed and were arranged in hexagonal array on the dorsal lingual surface. Capping each dermal papilla was a two-layered epithelium that protruded slightly above the lingual surface, thus forming the early filiform papilla. In the next stage of development, at 18-19 days of gestation, the epithelium lining the papilla had differentiated into two cell populations, one producing hard keratin, the other producing soft keratin. Some of the keratinized epithelial cells assumed a position at an acute angle to the tongue surface and extended deep into the epithelium. In the next stage, 20-21 days, a cleft appeared within these angularly oriented cells. This resulted in the division of the epithelium into keraatin-lined individual filiform papillae. Finally, the individual papillae increased in size to the adult form.     

The Expression of Beta ({beta}) Keratins in the Epidermal Appendages of Reptiles and Birds

The integuments of extant vertebrates display a variety of epidermalappendages whose patterns, morphology and terminal differentiation(epidermal keratins) depend upon interactions between ectodermal(epidermis) and mesodermal (dermis) tissues. In reptiles andbirds, appendage morphogenesis precedes terminal differentiation.Studies have demonstrated that appendage morphogenesis influencesthe expression of the appendage specific keratin genes. However,little is known about the nature of the structural genes expressedby the epidermal appendages of reptiles. How pattern formationand/or appendage morphogenesis influence terminal differentiationof reptilian appendages is not known. The epidermal appendages of reptiles and birds are characterizedby the presence of both alpha () and beta (ß) typekeratin proteins. Studies have focused on the genes of avianß keratins because they are the major structural proteinsof feathers. The occurrence of ß keratin proteinsin the scales and claws of both birds and reptiles and theirimmunological cross-reactivity suggest that the genes for reptilianß keratins may be homologous with those of birds.In bird appendages, the ß keratins are the productsof a large family of homologous genes. Specific members of thisgene family are expressed during the development of each appendage.Recent sequence analyses of feather ß keratins, fromdifferent orders of birds, demonstrate that there is more diversityat the DNA level than was implied by earlier protein sequencingstudies. Immunological techniques show that the same antibodies thatreact with the epidermal ß keratins of the chicken(Gallus domesticus) react with the epidermal ß keratinsof American alligators (Alligator mississippiensis). Furthermore,a peptide sequence (20 amino acids) from an alligator claw ßkeratin is similar to a highly conserved region of avian claw,scale, feather, and feather-like ß keratins. Theseobservations suggest that the ß keratin genes of avianepidermal appendages have homologues in the American alligator.Understanding the origin and evolution of the ß keratingene families in reptiles and birds will undoubtedly add toour understanding of the evolution of skin appendages such asscales and feathers.     

Isolation of taste buds from the foliate papillae of the rabbit

Summary A method to isolate taste buds from the foliate papillae of the rabbit tongue is described. The method comprises (a) separation of the epidermis from the dermal layer after treatment with dilute acetic acid, and (b) mechanical removal of the taste buds from the epithelium with the use of a surgical needle. The procedure yields taste buds that are morphologically well preserved, and in quantities sufficient to enable a detailed biochemical characterization. Preliminary tests have shown the taste buds to have biochemical properties clearly distinct from those of the adjacent epithelium. The method may provide a basis for studying the molecular mechanism of taste perception in greater detail.On leave of absence from the Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.