Molecular modelling of HLA-B*35:132.

Here we describe the molecular modelling of the new variant HLA-B*35:132. This allele shows one mismatch with B*35:01:01:01 in exon 3 at position 575 where a T is substituted by a C, which implies an amino acidic change from Leucine to Proline. This seems not to alter the molecular structure and not to compromise the HLA complex and T-cell receptor interaction.

Common and well-documented HLA alleles: 2012 update to the CWD catalogue.

We have updated the catalogue of common and well-documented (CWD) human leukocyte antigen (HLA) alleles to reflect current understanding of the prevalence of specific allele sequences. The original CWD catalogue designated 721 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, and -DPB1 loci in IMGT (IMmunoGeneTics)/HLA Database release 2.15.0 as being CWD. The updated CWD catalogue designates 1122 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 loci as being CWD, and represents 14.3% of the HLA alleles in IMGT/HLA Database release 3.9.0. In particular, we identified 415 of these alleles as being 'common' (having known frequencies) and 707 as being 'well-documented' on the basis of ~140,000 sequence-based typing observations and available HLA haplotype data. Using these allele prevalence data, we have also assigned CWD status to specific G and P designations. We identified 147/151 G groups and 290/415 P groups as being CWD. The CWD catalogue will be updated on a regular basis moving forward, and will incorporate changes to the IMGT/HLA Database as well as empirical data from the histocompatibility and immunogenetics community. This version 2.0.0 of the CWD catalogue is available online at cwd.immunogenomics.org, and will be integrated into the Allele Frequencies Net Database, the IMGT/HLA Database and National Marrow Donor Program's bioinformatics web pages.

16th IHIW: international histocompatibility working group in hematopoietic cell transplantation.

The International Histocompatibility Working Group is a collaborative international effort to understand the HLA and non-HLA genetics of the transplantation barrier. The Working Group is comprised of experts in the fields of histocompatibility and immunogenetics, hematopoietic cell transplantation and outcomes research. Data for 25 855 unrelated donor transplants were submitted in support of research studies for the 16th International Histocompatibility Workshop. Active investigation is in progress in seven key areas: the impact of HLA matching, role of race and ethnicity, identification of permissible HLA mismatches, haplotype-associated determinants, minor histocompatibility antigens, immune response genes and KIR genetics. New hypotheses for the 16th workshop were developed for immunogenetic studies in cord blood and haploidentical-related donor transplantation.

Genotypes and haplotypes in the 3' untranslated region of the HLA-G gene and their association with clinical outcome of hematopoietic stem cell transplantation for beta-thalassemia.

Polymorphisms in the 3' untranslated region (3'UTR) of HLA-G, an important player in immunological tolerance, could be involved in post-transcriptional expression control, and their association with different clinical immune-related conditions including autoimmunity and transplantation is of mounting interest. Most studies have focused on a 14 base pair (bp) insertion/deletion (ins/del), while additional single-nucleotide polymorphisms (SNPs) in the HLA-G 3'UTR have been described but not extensively investigated for their clinical relevance. Here we have comparatively studied the association between 3'UTR haplotypes of HLA-G, or the 14 bp ins/del, with clinical outcome of HLA-identical sibling hematopoietic stem cell transplantation (HSCT) in 147 Middle Eastern beta-thalassemia patients. Sequence based typing of 3'UTR HLA-G polymorphisms in the patients and in 102 healthy Italian blood donors showed strong linkage disequilibrium between the 14 bp ins/del and five 3'UTR SNPs, which together could be arranged into eight distinct haplotypes based on expectation-maximization studies, with four predominant haplotypes (UTRs1-4). After HSCT, we found a moderate though not significant association between the presence of UTR-2 in double dose and protection from acute graft versus host disease (hazard ratio (HR) 0.45, 95% confidence intervals (CI): 0.14-1.45; P = 0.18), an effect that was also seen when the corresponding 14 bp ins/ins genotype was considered alone (HR 0.42, 95% CI: 0.16-1.06; P = 0.07). No association was found with rejection or survival. Taken together, our data show that there is no apparent added value of considering entire 3'UTR HLA-G haplotypes for risk prediction after allogeneic HSCT for beta-thalassemia.

The peptide-binding motif for the human transporter associated with antigen processing.

Presentation of antigenic peptides by human leukocyte antigen class I molecules is dependent on peptide transport into the endoplasmic reticulum by the transporters associated with antigen processing (TAP) (Germain, R. N. 1994. Cell. 76:287-299). This translocation step is currently regarded as permissive for all peptides with COOH-terminal residues capable of binding to HLA class I molecules (Momburg, F., J. Roelse, J. C. Howard, G. W. Butcher, G.J. Hämmerling, and J.J. Neefjes. 1994. Nature (Lond.). 367:648-651). In this report, we show that the human transporter selects peptides according to a binding motif based on the strong effects on peptide affinity of the three NH2-terminal positions and the COOH-terminal residues. TAP favors strongly hydrophobic residues in position 3 (P3) and hydrophobic or charged residues in P2, whereas aromatic or acidic residues in P1, as well as Pro in P1 and P2, have strong deleterious effects. Selection of naturally presented peptides by the transporter is suggested by their higher average affinity for TAP, as compared to nonselected peptides. The TAP preferences in the three NH2-terminal positions correspond to those of the vast majority of human leukocyte antigen class I alleles, but they represent an obstacle for peptide supply to some alleles, e.g., the B7-like group. We propose that peptides binding to these alleles, and in general, peptides with TAP affinities below a certain threshold, may be transported as extended precursors.

High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals.

Using fluorescent HLA-A*0201 tetramers containing the immunodominant Melan-A/MART-1 (Melan-A) tumor-associated antigen (Ag), we previously observed that metastatic lymph nodes of melanoma patients contain high numbers of Ag-experienced Melan-A-specific cytolytic T lymphocytes (CTLs). In this paper, we enumerated and characterized ex vivo Melan-A-specific cells in peripheral blood samples from both melanoma patients and healthy individuals. High frequencies (>/=1 in 2,500 CD8(+) T cells) of Melan-A-specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals. Virtually all Melan-A-specific cells from 6 out of 6 healthy individuals and from 7 out of 10 patients displayed a naive CD45RA(hi)/RO(-) phenotype, whereas variable proportions of Ag-experienced CD45RA(lo)/RO(+) Melan-A-specific cells were observed in the remaining 3 patients. In contrast, ex vivo influenza matrix-specific CTLs from all individuals exhibited a CD45RA(lo)/RO(+) memory phenotype as expected. Ag specificity of tetramer-sorted A2/Melan-A(+) cells from healthy individuals was confirmed after mitogen-driven expansion. Likewise, functional limiting dilution analysis and interferon gamma ELISPOT assays independently confirmed that most of the Melan-A-specific cells were not Ag experienced. Thus, it appears that high frequencies of naive Melan-A-specific CD8(+) T cells can be found in a large proportion of HLA-A*0201(+) individuals. Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A-specific cells can occur in vivo.

Impact of HLA-G polymorphism on the outcome of allogeneic hematopoietic stem cell transplantation for metastatic renal cell carcinoma.

Renal cell carcinoma (RCC) is particularly sensitive to immune intervention. HLA-G, a non-classical HLA class I molecule with immunomodulatory properties, has been studied with regard to outcome after hematopoietic stem cell transplantation (HSCT), in particular the 14 bp insertion/deletion polymorphism in the 3' untranslated region. Here we analyzed n=56 patients affected by metastatic RCC who received an allogeneic HSCT between 1998 and 2006 in Milano, Marseille, Clermont-Ferrand and Stockholm. The 14 bp polymorphism was analyzed in correlation with overall survival (OS), PFS, acute and chronic GvHD. With a median follow-up of 13 years, a trend towards better outcome was observed when homozygosity for the 14bp-del allele was present: multivariate hazard ratio was 0.50 (95% confidence interval (CI): 0.23-1.13; P=0.10) and 0.57 (95% CI: 0.26-1.26; P=0.17) for OS and PFS, respectively, when 14bp-del/del was compared with 14bp-ins/X. Further exploratory analysis revealed a significant association between T/C at p3003 and improved OS (P=0.05) and PFS (P=0.006) compared with T/T. To our knowledge this is the first study on HLA-G and outcome after HSCT for a solid malignancy. After a coordinated multicenter study, we found that the more tolerogenic polymorphisms (14bp-del/del) is associated with better PFS and OS. The finding on p3003 deserves further investigation.

Directionality of non-permissive HLA-DPB1 T-cell epitope group mismatches does not improve clinical risk stratification in 8/8 matched unrelated donor hematopoietic cell transplantation.

In 8/8 HLA-matched unrelated donor (UD) hematopoietic cell transplants (HCT), HLA-DPB1 mismatches between alleles from different T-cell epitope (TCE) groups (non-permissive mismatches) are associated with significantly higher risks of mortality compared with those between alleles from the same TCE group (permissive mismatches); however, the relevance of mismatch directionality, that is (host vs graft (uni-directional HvG), graft vs host (uni-directional GvH) or both (bi-directional) in the non-permissive setting is unknown. We show here significantly higher in vitro relative responses (RR) to bi-directional mismatches compared with uni-directional HvG or GvH mismatches in a total of 420 one-way mixed lymphocyte reactions between 10/10 matched pairs (RR 27.5 vs 7.5 vs 15.5, respectively, P<0.001). However, in 3281 8/8 matched UD HCT for leukemia or myelodysplastic syndrome, the hazards of transplant-related mortality (TRM) were similar for uni-directional HvG or GvH mismatches and bi-directional mismatches (hazard ratio (HR) 1.32, P=0.001 vs HR 1.28, P=0.005 and HR 1.34, P=0.046), compared with permissive mismatches. Similar results were observed for overall survival. No statistical differences between the uni- and the bi-directional non-permissive groups were detected in pairwise comparisons for any of the outcomes tested. We conclude that consideration of directionality does not improve risk stratification by non-permissive HLA-DPB1 TCE mismatches in UD searches.

Common and well-documented HLA alleles over all of Europe and within European sub-regions: A catalogue from the European Federation for Immunogenetics.

BACKGROUND: A catalogue of common and well-documented (CWD) human leukocyte antigen (HLA), previously established by the American Society for Histocompatibility and Immunogenetics (ASHI), is widely used as indicator for typing ambiguities to be resolved in tissue transplantation or for checking the universality of any HLA allele in the world. However, European population samples, which are characterized by a substantial level of genetic variation, are underrepresented in the ASHI catalogue. Therefore, the Population Genetics Working Group of the European Federation for Immunogenetics (EFI) has facilitated data collection for an European CWD catalogue. MATERIALS AND METHODS: To this end, 2nd-field HLA-A, -B, -C,- DRB1,- DQA1,- DQB1 and -DPB1 data of 77 to 121 European population samples (21 571-3 966 984 individuals) from 3 large databases, HLA-net/Gene[VA], allelefrequencies.net and DKMS, were analysed. RESULTS: The total number of CWD alleles is similar in the EFI (N = 1048) and ASHI (N = 1031) catalogues, but the former counts less common (N = 236 vs 377) and more well-documented (N = 812 vs 654) alleles than the latter, possibly reflecting differences in sample numbers and sizes. Interestingly, approximately half of the CWD alleles reported by EFI were not reported by ASHI and vice-versa, underlining the distinct features of the two catalogues. Also, although 78 common alleles are widely distributed across Europe, some alleles are only common within specific sub-regions, showing regional variability. CONCLUSION: Although the definition of CWD alleles itself is affected by different parameters, calling for current updates of the list, the EFI CWD catalogue provides new insights into European population genetics and will be a very useful tool for tissue-typing laboratories in and beyond Europe.

Bone marrow mammaglobin expression as a marker of graft-versus-tumor effect after reduced-intensity allografting for advanced breast cancer.

We assessed mammaglobin (MMG) gene expression in bone marrow (BM) aspirates from patients with advanced breast cancer who had received a reduced-intensity conditioning and stem cell allografting, in order to detect a graft-versus-tumor effect on micrometastatic disease. Nine patients received a reduced-intensity conditioning with fludarabine, cyclophosphamide, and thiotepa, followed by peripheral blood allografting from HLA-identical sibling donors. Nested RT-PCR analysis with sequence-specific primers for MMG was carried out on a monthly basis on BM samples. Three patients had MMG-positive BM, four patients had MMG-negative BM before allografting, and two were undetermined. In two patients, a clinical response after allografting (partial remission) occurred concurrently with the clearance of MMG expression, at a median of 6 months after allografting, following immune manipulation. In two patients, a prolonged stable disease and negative MMG expression occurred after day +360 from allografting. In two patients, progression of the disease was associated with MMG RT-PCR changing from negative to positive. In one case, a disease response occurring after donor lymphocyte infusion and grade II acute GVHD was heralded by negativization of MMG expression. Although preliminary, these data suggest that a graft-versus-breast cancer effect is detectable on micrometastatic BM disease.

The DAM gene family encodes a new group of tumor-specific antigens recognized by human leukocyte antigen A2-restricted cytotoxic T lymphocytes.

The DAM family of genes has a high degree of homology with MAGE, both in nucleotide sequence and in neoplastic tissue-specific expression. This study describes, for the first time, the identification of CTLs specific for a peptide epitope encoded by DAM genes. A human leukocyte antigen (HLA)-A2-restricted CTL clone was raised against a peptide, D10/6-271, encoded by codons 271-279 in the DAM cDNA. The corresponding peptide in the MAGE-3 sequence, M3-271, has been shown previously to be a natural T-cell epitope for HLA-A2-restricted CTLs recognizing the MAGE-3 protein. The D10/6-271-specific CTL clone required approximately 3 nM exogenous peptide for half-maximal lysis of target cells and was able to specifically recognize endogenous DAM antigen on HLA-A2+ melanoma cells infected with a vaccinia vector recombinant for gene DAM-6. These data suggest that DAM genes might encode a new group of tumor-specific antigens useful for the design of specific antitumor vaccines.

YKL-40 in allogeneic hematopoietic cell transplantation after AML and myelodysplastic syndrome.

YKL-40, also called chitinase-3-like-1 protein, is an inflammatory biomarker that has been associated with disease severity in inflammatory and malignant diseases, including AML, multiple myeloma and lymphomas. The objective of the current study was to assess the prognostic value of pretransplant recipient and donor plasma YKL-40 concentrations in patients with AML (n=624) or myelodysplastic syndrome (n=157) treated with allogeneic hematopoietic cell transplantation (HCT). In recipients, the plasma YKL-40 concentrations were increased when the HCT-comorbidity index was ⩾5 (P=0.028). There were no significant associations between plasma YKL-40 concentrations in recipients and any outcome measures. In donors with YKL-40 plasma concentrations above the age-adjusted 95th percentile, a trend toward increased grade II-IV acute GvHD in recipients was observed (adjusted hazard ratio 1.39 (95% confidence interval 1.00-1.94), P=0.050), with no significant associations with overall survival, treatment-related mortality or relapse. In conclusion, our study shows that YKL-40 does not aid risk stratification of patients undergoing allogeneic HCT, but suggests that YKL-40 may aid donor selection when multiple, otherwise equal, donors are available.

HLA class I in acute promyelocytic leukemia (APL): possible correlation with clinical outcome.

The majority of patients with acute promyelocytic leukemia (APL) possess either a bcr1 or a bcr3 type fusion between PML and RARalpha genes. The junction sequences may possibly be a target for immune response and influence susceptibility to the disease. In this case, HLA class I allele frequencies would be different between bcr1 and bcr3 patients. To test this hypothesis, we typed 102 APL patients for HLA-A, -B and -Cw alleles. The A*1, A*30, B*51, B*41, Cw*0602, and Cw*1701 alleles showed a different distribution between bcr1 and bcr3 patients, but in no case was this statistically significant after correction for the number of comparisons or was confirmed in an independent panel. Moreover, no difference was detected between bcr1 and bcr3 when HLA alleles were grouped according to their peptide binding specificities. Comparing HLA frequencies, clinical features at diagnosis and clinical outcome of the 64 patients homogeneously treated with all-trans retinoic acid and idarubicin (AIDA protocol) we observed a statistically significant association between HLA-B*13 and risk of relapse by univariate and multivariate regression analysis. Should this finding be confirmed in larger future studies, this observation would be of outmost importance in identifying patients at high risk of relapse in which more aggressive consolidation therapies should be used.

Incidence, risk factors and clinical outcome of leukemia relapses with loss of the mismatched HLA after partially incompatible hematopoietic stem cell transplantation.

Genomic loss of the mismatched human leukocyte antigen (HLA) is a recently described mechanism of leukemia immune escape and relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Here we first evaluated its incidence, risk factors and outcome in 233 consecutive transplants from partially HLA-mismatched related and unrelated donors (MMRD and MMUD, respectively). We documented 84 relapses, 23 of which with HLA loss. All the HLA loss relapses occurred after MMRD HSCT, and 20/23 in patients with acute myeloid leukemia. Upon MMRD HSCT, HLA loss variants accounted for 33% of the relapses (23/69), occurring later than their 'classical' counterparts (median: 307 vs 88 days, P<0.0001). Active disease at HSCT increased the risk of HLA loss (hazard ratio (HR): 10.16; confidence interval (CI): 2.65-38.92; P=0.001), whereas older patient ages had a protective role (HR: 0.16; CI: 0.05-0.46; P=0.001). A weaker association with HLA loss was observed for graft T-cell dose and occurrence of chronic graft-versus-host disease. Outcome after 'classical' and HLA loss relapses was similarly poor, and second transplantation from a different donor appeared to provide a slight advantage for survival. In conclusion, HLA loss is a frequent mechanism of evasion from T-cell alloreactivity and relapse in patients with myeloid malignancies transplanted from MMRDs, warranting routine screening in this transplantation setting.

Sirolimus-based graft-versus-host disease prophylaxis promotes the in vivo expansion of regulatory T cells and permits peripheral blood stem cell transplantation from haploidentical donors.

Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients' bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II-IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transpant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus-mycophenolate-ATG-F-rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts.

Profound biotinidase deficiency in two asymptomatic adults.

Biotinidase deficiency is an autosomal-recessive disorder of biotin recycling. Children with profound biotinidase deficiency usually have neurological and cutaneous symptoms in early childhood, but they may not develop symptoms until adolescence. We now report on a man and a woman with profound biotinidase deficiency who are asymptomatic and who were diagnosed only because their biotinidase-deficient children were identified by newborn screening. These adults have never exhibited symptoms of the disorder and are homozygous for two different mutations resulting in different aberrant enzymes. There is no evidence of an increased dietary intake of biotin to explain why they have remained asymptomatic. Although these adults may still be at risk for developing symptoms, they could represent a small group of individuals with profound biotinidase deficiency who will never develop clinical problems. Their lack of symptoms suggests that there are probably epigenetic factors that protect some enzyme-deficient individuals from developing symptoms. These individuals broaden the spectrum of expression of biotinidase deficiency.

Biotinylation of biotinidase following incubation with biocytin.

Human serum biotinidase, purified to homogeneity (1920 units/mg protein), was incubated with biocytin prior to electrophoresis and transblotting with avidin-peroxidase. Avidin reacted with biotinidase maximally when incubated at pH 7.5-9, less at pH 7 and none below pH 7. No avidin reactivity occurred when biotinidase was incubated with biotin or in the absence of biocytin. Inclusion of the nucleophilic acceptors, ethanolamine or hydroxylamine, to the incubation mixture with biocytin and biotinidase resulted in loss of avidin reactivity. High concentrations of mercaptoethanol also prevented avidin reactivity. These results suggest that biotinidase can be biotinylated in the presence of biocytin at neutral to alkaline pH probably through a thioester bond formed with a cysteine residue in the active site of the enzyme. Biotinidase may then function as a biotinylating enzyme when incubated with appropriate nucleophilic acceptors.

Morphological characteristics of neocortical laminae when studied in tangential semithin sections through the visual cortex of the rabbit.

In semithin sections cut tangentially with respect to the surface of the cerebral cortex and with a side length of at most a few mm, it is extremely difficult to be certain which lamina has been sectioned, for instance lamina III or upper lamina IV. In order to provide criteria to overcome this difficulty, a systematic study was carried out in which 1 micron semithin sections through the visual cortex of the rabbit were cut in both the frontal and tangential planes and compared with 8-12 micron paraffin sections stained for cells and/or myelin. It was found that the arrangement of thick dendrites, of myelinated fibres and of unmyelinated profiles which may be either dendrites or neurites, permits subdivision of the visual cortex into three characteristic horizontal zones which are termed zones A, B and C. The relation between these three zones and the cytoarchitectonic pattern is as follows: Zone A corresponds to the cytoarchitectonic lamina I; zone B comprises lamina II/III, lamina IV and the upper half of lamina V; zone C corresponds to the lower half of lamina V and all of lamina VI. Zones A and B can further be subdivided: Zone A consists of two layers, whereas zone B can be divided into three tiers, each of which is characterized by a particular arrangement of the myelinated and unmyelinated profiles.(ABSTRACT TRUNCATED AT 250 WORDS)

A pattern formed by preferential orientation of tangential fibres in layer I of the rabbit's cerebral cortex.

1. The tangential organization of layer I has been studied in frozen sections impregnated according to a modified Liesegang method and in Bodian impregnated paraffin sections cut tangentially to the dorsal surface of the rabbit's cerebral cortex. 2. It is shown that sublamina tangentialis of layer I contains a system of parallel nerve fibres forming a distinct pattern in the tangential plane. 3. This pattern has been reconstructed for a large region of the dorsal surface of the cerebral cortex including the striate areas as well as the peristriate, parietal and precentral agranular regions and parts of the retrosplenial area. 4. In most parts of the region investigated, the tangential fibres of layer I are oriented in an antero-medial to postero-lateral direction, forming an angle of about 50 degrees with the sagittal plane. 5. Deviations from this pattern are found in the furrows formed by the lateral sulcus and the frontal impression and also in the caudal part of the retrosplenial area. In these regions, which are characterized by comparatively steep changes of the cortical relief, the fibres course in a more sagittal direction.

Postnatal growth of the dorsal lateral geniculate nucleus of the cat.

The postnatal growth of the dorsal part of the lateral geniculate nucleus (LGNd) is studied in paraffin sections through the brains of 32 cats of known age. The changes in shape and position of the LGNd are described and it is shown that its volume increases from about 3.4 mm3 at birth to about 26.4 mm3 in the adult cat. When this value is corrected for shrinkage, the volume of the LGNd in the adult cat turns out to be about 44 mm3. The detailed measurements reveal that during the second and third week of postnatal life there is a particularly steep increase in volume and that the final values are already reached at around the 40th day. Concomitant with the increase in volume there is a decrease of the number of cells per unit volume of grey matter. In the binocular segment of lamina A the number of cells decreases from about 470 per (0.1 mm)3 at birth to between 95 and 130 per (0.1 mm)3 in the adult cat. Separate measurements of nerve cells and neuroglial cells indicate that the absolute number of nerve cells remains fairly constant during postnatal life, whereas between the second and sixth week a great number of neuroglial cells are newly formed.