EXPERIMENTAL ANTI-GBM GLOMERULONEPHRITIS INDUCED IN RATS BY IMMUNIZATION WITH SYNTHETIC PEPTIDES BASED ON SIX α CHAINS OF HUMAN TYPE IV COLLAGEN

The anti-glomerular basement membrane (GBM)-nephritis-inducing activity of six synthetic peptides having an amino acid sequence consisting of the six α chains of human type IV collagen was examined by injecting the peptides into rats. The peptides consisted of 27 amino acid residues from the non-collagenous domain (NC1) of the α1 to α6 chains and were non-consensus sequences sandwiched between two consensus sequences near the carboxyl terminus. Each peptide was coupled to keyhole limpet haemocyanin and injected with adjuvant into the footpads of 20 female WKY–Crj rats. The number of rats with proteinuria (over 10·0 mg of urinary protein/15 h) and haematuria was 2 with the α3 peptide, 8 with the α4 peptide, and 1 with the α5 peptide. Histological changes seen in the glomeruli were characteristic of those in anti-GBM nephritis. Linear deposition of rat IgG along the GBM was observed in five rats injected with the α4 peptide. A nephritogenic monoclonal antibody against the α4 peptide was established using lymph node cells from a rat injected with the α4 peptide. The results indicate that α4(IV)NC1 is a potent nephritogenic antigen like α3(IV)NC1, which has already been recognized as a primary target antigen in Goodpasture's syndrome.     

COLLAGEN DISTRIBUTION IN FOCAL AND SEGMENTAL GLOMERULOSCLEROSIS: AN IMMUNOFLUORESCENCE AND ULTRASTRUCTURAL IMMUNOGOLD STUDY

Focal and segmental glomerulosclerosis (FSGS) is a non-specific scarring process of the glomerulus, initially described in idiopathic nephrotic syndrome. The distribution of types I, III, IV, V, and VI collagen and of the α 1, α 3, α 4, α 5, and α6 chains of type IV collagen was studied by immunohistochemistry in sclerotic lesions of nine nephrotic children. Dual immunofluorescence and high-resolution immunogold labelling were used to determine the precise distribution of the antigens. No changes were detected in normal glomeruli of patients compared with controls. In FSGS, type IV collagen [α 1(IV)2 α2(IV)], and to a lesser degree type VI, accumulates in the two components of the lesion: the enlarged mesangial matrix and the material deposited between the pushed-out podocytes and the α 3– α 5(IV)-positive glomerular basement membrane. Staining for α6(IV) and types I, III, and V collagen was practically negative. These results suggest that the matrix components of the sclerotic lesion are produced solely by glomerular cells. Changes in the relative distribution of type IV collagen chains, characterized by the presence of collagen [α 1(IV)2 α2(IV)] in close contact with the podocytes, strongly suggest a switch in the podocyte programme of collagen synthesis.     

Recombinant α-chains of type IV collagen demonstrate that the amino terminal of the Goodpasture autoantigen is crucial for antibody recognition

Goodpasture's disease, an autoimmune disorder causing severe glomerulonephritis and pulmonary haemorrhage, is characterized by antibodies to the glomerular basement membrane (GBM). The principal target antigen has been identified as the carboxyl terminal non-collagenous (NC1) domain of the α3-chain of type IV collagen. Anti-GBM antibodies appear to recognize one major epitope that is common to all patients, and is largely conformational. We have analysed antibody binding to recombinant α(IV)NC1 domains using a construct and expression system shown to produce correctly folded antigen that is strongly recognized by autoantibodies. In this system, as with the native antigen, α3(IV)NC1 was bound strongly by antibodies from all patients, whereas the closely related α1(IV) and α5(IV)NC1 domains, similarly expressed, showed no such binding. A series of chimeric NC1 domains, between human α3(IV) and α1(IV), and between human and rat α3(IV), were expressed as recombinant molecules, and were recognized by autoantibodies to varying degrees. Strong binding required the presence of human α3(IV) sequence in the amino terminal region of both sets of chimeric molecules. This work strongly suggests that the amino terminal of α3(IV)NC1 is critical for antibody recognition, whereas the carboxyl terminal end of α3(IV)NC1 has a less important role.     

Differential distribution of basement membrane type IV collagen alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains in colorectal epithelial tumors

In this study, we examined the relationship between the histopathological grade and immunohistochemical localization of six genetically distinct type IV collagen alpha chains, the major component of basement membrane (BM), in normal and neoplastic colorectal tissues. In the normal colorectal mucosa, alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were stained in all epithelial BM. However, alpha3/alpha4(IV) chains were restrictively immunostained in the BM of the apical surface epithelium. Similar immunostaining profiles for alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were observed in tubular adenomas with mild/moderate atypia. However, in intramucosal carcinomas, both alpha1/alpha2(IV) chains were linearly stained in the BM of cancer cell nests, while the assembly of alpha5/alpha6(IV) chains into the BM was inhibited in a discontinuous or negatively stained pattern. The normal colorectal mucosa forms a second network of BM composed of alpha5/alpha6(IV), partly alpha3/alpha4(IV) chains, in addition to the classic network of alpha1/alpha2(IV) chains. The differential immunohistochemical localization of the type IV collagen alpha5/alpha6 chains could be one diagnostic marker for the invasiveness of colorectal cancer.     

The human embryo produces basement membrane collagen (type IV collagen)-degrading protease activity

The biochemical basis and mechanism of embryo implementationis poorly understood. The human embryo has to penetrate theendometrial basement membrane and the thick decidual wall duringimplantation, a process which resembles the active spreadingof invasive tumour cells where the degradation of type IV collagen(basement membrane collagen) plays an important role. This studyreports that human embryos produce type IV collagen-degradingenzyme activity and the secretion of this enzyme increases withtime in culture. The type IV collagen-degrading enzyme activitymight facilitate the penetration of the embryo through the decidua,thus emphasizing an active role of the embryo in implantation.On the other hand, unfertilized oocytes secrete low, stableamounts of type IV collagen-degrading enzyme activity in vitro.It was also found that follicular granulosa cells secrete highamounts of type IV collagenolytic activity in culture. It hasbeen previously shown that there is a pre-ovulatory peak intype IV collagenolytic activity in follicular fluid, and itcan be assumed that the appearance of this enzyme in the follicularfluid is probably connected to follicular rupture and that itis produced by granulosa cells     

Collagen type Ⅳ对周围神经中再生轴突及非神经元细胞的作用和影响

本文用抗ｃｏｌｌａｇｅｎｔｙｐｅⅣ对抗体阻断ｃｏｌｌａｇｅｎｔｙｎｅⅣ的方法，研究了ｃｏｌｌａｇｅｎｔｙｐｅⅣ失活的移植神经段（长１０ｍｍ）植入大鼠坐骨神经后对再生轴突和非神经元细胞的作用和影响．实验结果显示：在移植神经段近端距近侧吻合口１ｍｍ处，术后１０ｄ抗ｃｏｌｌａｇｅｎｔｙｐｅⅣ组再生轴突数为对照组的５４％，术后１５ｄ增加到６６％，术后３０ｄ高达９４％．在移植神经段远侧距近侧吻合口９ｍｍ处，术后３０ｄ抗ｃｏｌｌａｇｅｎｔｙｐｅⅣ组再生轴突数为对照组的５８％。表明抗ｃｏｌｌａｇｅｎｔｙｐｅⅣ组再生轴突的生长启动和生长速度明显慢于对照组．巨噬细胞在移植神经段内的滞留数量抗ｃｏｌｌａｇｅｎｔｙｐｅⅣ组明显多于对照组．这些结果揭示ｃｏｌｌａｇｅｎｔｙｐｅⅣ在神经损伤和再生中对促进轴突的生长和维持神经微环境的平衡起着积极的作用．本文对ｃｏｌｌａｇｅｎｔｙｐｅⅣ在神经再生中的作用机制作了初步的分析和探讨。     

A comparison of antibodies to type VII and type IV collagen laminin and amnion as epidermal basement membrane markers

We have compared four monoclonal antibodies which label basement membrane components using an indirect immunoperoxidase method in frozen sections of skin biopsies. The antibodies LH 7.2 and GB3 showed expression limited to epidermal and adnexal basement membrane. Antibodies to laminin and type IV collagen also decorated dermal blood vessels and stromal components. The antibodies LH 7.2 and GB3 are more suitable for labelling epidermal basement membrane in the study of cutaneous lesions.     

Loss of α6 and β4 integrin subunits coincides with loss of basement membrane components in oral squamous cell carcinomas

In oral squamous cell carcinomas, focal or extensive loss of basement membrane components and of integrins has been reported. The purpose of this study was to investigate whether those regions of the tumour-connective tissue interface which lack laminin and type IV collagen coincide with areas of loss of the α₆ and β₄ integrin subunits on basal keratinocytes. Out of a total of 15 poor and moderately or well differentiated squamous cell carcinomas, all showed some loss or fragmentation of basement membrane proteins and in 12 the loss was coincident with loss of α₆ and/or β₄. In three cases, there was loss of basal integrin expression in areas where the basement membrane remained intact. These results provide further evidence that loss of integrins may play an important role in tumour progression and prompt us to speculate about the sequence of events leading to tumour invasion.     

Ultrastructural triple localization of laminin‐1, nidogen‐1, and collagen type IV helps elucidate basement membrane structure in vivo

The basement membrane models which have been proposed to date are generally based on biochemical data, mainly binding studies and artificially synthesized polymers in vitro. Basically these have led to models proposing two three‐dimensional laminin‐1 and collagen type IV networks interconnected by nidogen‐1. Whether they reflect the in vivo basement membrane structure is still not clear. We localized laminin‐1, nidogen‐1, and collagen type IV ultrastructurally in adult and fetal mouse kidney basement membranes with the help of immunogold‐histochemistry performing double and triple localization to try to elucidate the molecular organization of basement membranes in vivo. We found laminin‐1, nidogen‐1, and collagen type IV distributed over the entire basement membranes in adult and fetal kidneys. This contradicts earlier studies ascribing laminin‐1 to the lamina lucida and collagen type IV to the lamina densa. In addition, various basement membrane segments exhibited an organized labeling pattern for the BM components. Double‐labeling revealed co‐localization of laminin‐1 and nidogen‐1. We conclude that the combination of laminin‐1 with collagen type IV as double‐network basement membrane partially interconnected by nidogen‐1 is found already in the early fetal kidney in vivo. However, our data cannot exclude the possibility of other variants of basement membrane assemblages. This is also indicated by a changing structure even in individual segments of one basement membrane type which renders a more flexible basement membrane architecture plausible. Anat Rec 254:382–388, 1999. © 1999 Wiley‐Liss, Inc.     

Basement membrane deposition in benign and malignant naevo-melanocytic lesions: an immunohistochemical study with antibodies to type IV collagen and laminin

Patterns of basement membrane deposition were investigated in benign and malignant naevo-melanocytic lesions using antibodies to type IV collagen and laminin. Paraffin sections required pretreatment with 6 M guanidine-HCl in addition to pepsin pretreatment. Basement membrane deposition was found around clusters as well as individual naevo-melanocytic cells in contact with dermal stroma. However, between keratinocytes and intra-epidermally located naevo-melanocytic cells, basement membrane immunostaining could not be detected. Tumour cell-stromal interaction is apparently a prerequisite for basement membrane deposition in naevo-melanocytic lesions. Basement membrane discontinuities, in the absence of inflammatory infiltrate, appeared, in doubtful cases, to be evidence in favour of malignant melanoma. The general pattern of basement membrane deposition in benign and malignant lesions was found to be similar and therefore of no help in differential diagnosis. Identification of hyaline bodies, which show immunoreactivity with antibodies to basement membrane components, may be helpful in distinguishing between Spitz naevi and malignant melanomas. Detection of vascular invasion, a prognostic indicator in malignant melanoma, is facilitated by basement membrane immunostaining.     

The distribution of type IV collagen in invasive carcinoma of the uterine cervix

The presence of type IV collagen at the tumour/stromal interface of invasive carcinoma of the cervix was evaluated in 60 cases. It was assessed semi-quantitatively according to its staining characteristics, and expressed as: thick, with or without minimal discontinuity; thin, with or without moderate discontinuity; fragmentary or absent. In each case the tumour type was identified, the differentiation, growth pattern and peritumoural inflammatory infiltrate was graded and lymph node status established. There was, overall, a significant correlation between a paucity of type IV collagen and a poorer prognosis and an infiltrating growth pattern. There was a highly significant correlation for adenocarcinomas in relation to poor outcome, infiltrating growth pattern and lymph node metastases. By contrast, no statistically significant correlation was found between paucity of type IV collagen and the other parameters for either squamous or adenosquamous carcinomas. The presence of absence of type IV collagen may be a useful prognostic marker, particularly for adenocarcinomas, and may play a part in the invasive and metastatic process.     

INTENSIFIED IgA MESANGIAL DEPOSITS AFTER ADMINISTRATION OF SHEEP ANTI-TYPE IV COLLAGEN SERUM IN MICE

Sheep anti-type IV collagen serum was intravenously administered to male mice of the BALB/c, C3H and ddY strains, and their kidneys were morphologically studied monthly for 10 months thereafter. By immunofluorescence, the sheep IgG was seen to have immediately become conjugated to the glomeruli, mainly in a mesangial pattern. Successively, autologous mouse C3 and IgG appeared with the same type of distribution. Within 3 to 4 months after the start of the experiment, mouse IgA also appeared in the mesangium, especially in ddY mice. The intensity and frequency of mesangial IgA deposition and the serum IgA level increased with time in this strain. BALB/c and C3H mice also showed the same tendency of mesangial IgA deposition, although to a lesser degree. In summary, it was concluded that mesangial IgA deposition was due to non-immunological local trapping, on the basis of the results obtained by ELISA analysis of the sera and renal eluate. Although the ddY mouse is known to show spontaneous mesangial IgA deposition associated with a high serum IgA level with aging, these characteristics were much accelerated and intensified by this antiserum treatment. The relation of this observation to the pathogenesis of human IgA nephritis is discussed. ACTA PATHOL JPN 38: 141–152, 1988.     

PROGNOSTIC VALUE OF MIB-1 IN NEUROENDOCRINE TUMOURS OF THE LUNG

The spectrum of neuroendocrine lung tumours ranges from highly aggressive small cell carcinomas (SCLC) to carcinoid tumours (CD) of low malignant potential. Between these two extremes, the ‘well-differentiated neuroendocrine carcinomas’ (WDNEC) form a transitional group with uncertain biological behaviour. This study investigated the prognostic value of the proliferation marker MIB-1 (paraffin Ki-67) in 59 neuroendocrine lung tumours (32 SCLC, 13 WDNEC, 14 CD) by immunostaining of routinely processed paraffin sections. Morphometric evaluation was done by semi-automatic image analysis. The results were compared with survival data (mean follow-up: 42 months). The proliferation rates of the tumours as determined by MIB-1 immunoreactivity (MIB-1-PR) were significantly different between the tumour types (SCLC>WDNEC>CD) and showed a strong inverse correlation with survival time. In CD, the percentage of MIB-1-labelled nuclei never exceeded 1·1 per cent; higher values would therefore favour the diagnosis of WDNEC over that of CD. Among WDNEC, MIB-1 was able to differentiate a subgroup with excellent prognosis (MIB-1-PR: 0·3–3·4 per cent) from another subgroup with a death rate of 50 per cent (MIB-1-PR: 7·3–20·3 per cent). Within each tumour type, all patients without distant metastases at diagnosis survived when MIB-1-PR was ⩽9·4 per cent, suggesting a potential threshold for prognosis. Although the status of metastases was the dominant prognostic factor in these neoplasms, MIB-1 was able to provide additional prognostic information allowing the definition of prognostically different subgroups of patients. In conclusion, MIB-1 and the status of metastases are complementary prognostic indicators and are best used in combination to characterize the biological behaviour of neuroendocrine lung tumours.     

MICROVASCULAR ANGIOPATHY IN ADVANCED PERIODONTAL DISEASE

Previous studies have shown a perivascular hyaline thickening affecting restricted regions of the microcirculation in gingivitis and moderate periodontitis and in the pulpal vessels in chronic pulpitis. In the present study of the lesion of advanced periodontitis, immunostaining for type IV collagen and laminin demonstrated widespread deposition of basement membrane material, with manifest involvement of the venous network. Some vessels were associated with an increased deposition of both basement membrane proteins, while others showed preferential deposition of either laminin or type IV collagen. Immunostaining also revealed an extensive trabecular network of type IV collagen throughout the affected gingival tissue that was not related to recognizable vessels but was co-extensive with less intense staining for laminin. This network was not associated with viable endothelial cells demonstrable by staining with the endothelial marker Ulex agglutinin (UEA-1). The results indicate extensive vascular pathology in advanced periodontitis that could explain the attenuation of the inflammatory reaction and the restricted ability to develop reparative granulation tissue in this disease.     

LUNG TUMOURS IMMUNOREACTIVE FOR PARATHYROID HORMONE RELATED PEPTIDE: ANALYSIS OF SERUM CALCIUM LEVELS AND TUMOUR TYPE

Secretion by tumours of parathyroid hormone-related peptide (PTHrP) in quantities sufficient to raise circulating levels results in the syndrome of humoral hypercalcaemia of malignancy (HHM). Since HHM is commonly associated with squamous carcinoma of lung and rarely with adenocarcinoma or lung neuroendocrine tumours, immunopositivity was related to tumour type, to assess whether this difference was due to a low general incidence of PTHrP expression in the latter two groups. Seventy-six of 82 tumours were immunopositive: 22 of 22 squamous carcinomas, 21 of 25 small cell lung carcinomas, 14 of 15 carcinoids, and 19 of 20 adenocarcinomas. These data confirm and extend previous observations on squamous and neuroendocrine tumours but are in contrast with previous findings in adenocarcinoma, which have suggested that only a small proportion of cases express the peptide. They suggest that the differences in incidence of HHM in the various tumour types are due to patterns of secretion, rather than differences in expression of PTHrP. The second aim of this study was therefore to assess whether tumours immunopositive for PTHrP, but not associated with HHM, might secrete PTHrP at levels which might result in more subtle changes in calcium metabolism. Preoperative calcium levels were analysed in a series of 56 patients with immunopositive lung tumours of all types. One small cell carcinoma was associated with hypercalcaemia, but there was no evidence of any other alteration in serum calcium. These data indicate that the majority of tumours expressing PTHrP do not secrete it in amounts sufficient to alter calcium metabolism.     

人前磨牙牙髓牙本质复合体Ⅰ型和Ⅲ型胶原的来源及分布

目的 探讨牙髓牙本质复合体Ⅰ型和Ⅲ型胶原的来源与分布。方法 收集人前磨牙，固定、脱钙、石蜡包埋、切片、用SABC法进行免疫组织化学染色，显示Ⅰ型和Ⅲ型胶原蛋白。结果Ⅰ型胶原在牙髓内十分丰富，聚集成束，根髓中致密，以粗纤维束沿神经血管纵行分布；冠髓中纤维束较细、大量分支、变细，随意分布，交织成疏松的纤维网。从根尖至牙冠，从牙髓中央至周边，纤维束由粗变细，沿途分支，大部分终止于牙髓基质，少部分经成牙本质细胞间进入前期牙本质。此外成牙本质细胞、成纤维细胞与血管内皮细胞亦见Ⅰ型胶原蛋白表达。前期牙本质Ⅰ型胶原染色深，与牙髓基质和成牙本质细胞突起内者相延续，成熟牙本质近髓1／3可见Ⅰ型胶原阳性免疫反应物。Ⅲ型胶原在牙髓牙本质复合体的表达基本与Ⅰ型原类似，仅数量和密度略少。结论 牙髓牙本质复合体内的Ⅰ型和Ⅲ型胶原可能由成牙本质细胞、成纤维细胞和血管内皮细胞产生；不仅牙髓内存在Ⅰ型和Ⅲ型胶原，而且前期牙本质也有Ⅰ型和Ⅲ型胶原。     

Loss of alveolar basement membrane type IV collagen alpha3, alpha4, and alpha5 chains in bronchioloalveolar carcinoma of the lung

Type IV collagen, the major component of basement membrane (BM), is composed of six genetically distinct alpha(IV) chains. This study investigated for the first time the expression of these six alpha(IV) chains immunohistochemically, using alpha(IV) chain-specific monoclonal antibodies, in normal lung and in small (less than 2 cm in diameter) adenocarcinoma of the lung with a bronchioloalveolar growth pattern at the periphery. Small adenocarcinomas were histopathologically classified into three subtypes: bronchioloalveolar carcinoma (BAC) without collapse, BAC with collapse, and adenocarcinoma with bronchioloalveolar features. In normal lung, alveolar BM was composed of alpha1(IV)/alpha2(IV) chains and alpha3(IV)/alpha4(IV)/alpha5(IV) chains. In non-collapsed areas of BAC, alveolar BM was composed of linear alpha1(IV)/alpha2(IV) chains and discontinuous alpha3(IV)/alpha4(IV)/alpha5(IV) chains. In collapsed areas of BAC, alveolar BM was composed of linear and thick alpha1(IV)/alpha2(IV) chains only, because of the complete loss of alpha3(IV)/alpha4(IV)/alpha5(IV) chains. In invasive areas of adenocarcinoma with bronchioloalveolar features, alpha1(IV)/alpha2(IV) chains around the cancer cell nests were disrupted, in addition to the complete loss of alpha3(IV)/alpha4(IV)/alpha5(IV) chains. In conclusion, during the process of stromal invasion of lung adenocarcinoma, type IV collagen of alveolar BM is remodelled from the complete type, composed of alpha1(IV)/alpha2(IV)/alpha3(IV)/alpha4(IV)/alpha5(IV) chains, to the incomplete type, composed of only alpha1(IV)/alpha2(IV) chains, before the disruption of alpha1(IV)/alpha2(IV) chains. These findings may help to clarify the molecular mechanisms of cancer invasion.