CPAMD8 loss-of-function underlies non-dominant congenital glaucoma with variable anterior segment dysgenesis and abnormal extracellular matrix.

Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG. CPAMD8 is a gene of unknown function and recently associated with ASD. Bioinformatic and in vitro functional evaluation of the variants using quantitative reverse transcription PCR and minigene analysis supported a loss-of-function pathogenic mechanism. Optical and electron microscopy of the trabeculectomy specimen from one of the CG-ASD cases revealed an abnormal anterior chamber angle, with altered extracellular matrix, and apoptotic trabecular meshwork cells. The CPAMD8 protein was immunodetected in adult human ocular fluids and anterior segment tissues involved in glaucoma and ASD (i.e., aqueous humor, non-pigmented ciliary epithelium, and iris muscles), as well as in periocular mesenchyme-like cells of zebrafish embryos. CRISPR/Cas9 disruption of this gene in F0 zebrafish embryos (96 hpf) resulted in varying degrees of gross developmental abnormalities, including microphthalmia, pharyngeal maldevelopment, and pericardial and periocular edemas. Optical and electron microscopy examination of these embryos showed iridocorneal angle hypoplasia (characterized by altered iris stroma cells, reduced anterior chamber, and collagen disorganized corneal stroma extracellular matrix), recapitulating some patients' features. Our data support the notion that CPAMD8 loss-of-function underlies a spectrum of recessive CG-ASD phenotypes associated with extracellular matrix disorganization and provide new insights into the normal and disease roles of this gene.

Changes of proteases and proteinase inhibitors in androgen-dependent advanced prostate cancer patients with alpha2-macroglobulin deficiency.

BACKGROUND: It is thought that the quantitative imbalance between proteases and their inhibitors is a causative factor in invasion and metastasis of cancer cells. We previously reported on a number of androgen-dependent advanced prostate cancer (PCa) patients in which serum alpha2-macroglobulin (alpha2M) levels were markedly decreased to < 20 mg/dL (defined as alpha2M deficiency). Anti-androgen therapy is at first generally very effective for androgen-dependent advanced PCa, yielding survival benefits for most patients. In the present study, we evaluated serum levels of PSA, matrix metalloproteinases-2 (MMP-2), alpha2M, and alpha2-plasmin inhibitor (alpha2PI) in advanced PCa patients with or without alpha2M deficiency in order to determine the clinical significance of these proteases and proteinase inhibitors for PCa progression. METHODS: In this study, 33 PCa patients were diagnosed at the Kitasato University Hospital and compared with 10 healthy controls. PSA and MMP-2 levels were determined by enzyme immunoassay. Measurement of alpha2M was performed by laser-nephelometry, alpha2PI levels were determined by turbidimetric immunoassay. RESULTS: Serum levels of PSA and MMP-2 in PCa patients with alpha2M deficiency were significantly higher than in patients not alpha2M-deficient. In contrast, serum levels of alpha2M and alpha2PI in these patients were significantly lower than in those not alpha2M-deficient. PSA and alpha2M levels showed an inverse relationship in androgen-dependent advanced PCa with alpha2M deficiency. CONCLUSIONS: Our findings indicate that the serum levels of these proteases and proteinase inhibitors, which are involved in the invasion and metastasis of PCa, may be indicators of PCa disease progression in addition to PSA levels.

Clinicopathological characteristics of androgen-dependent advanced prostate cancer patients with α2-macroglobulin deficiency.

α2-Macroglobulin (α2M) is thought to be involved in cancer metastasis and inflammatory reaction through its functions as a proteinase inhibitor and carrier protein for interleukin-6 (IL-6). We previously reported that advanced prostate cancer (PCa) patients with multiple distant bone metastases had markedly decreased serum α2M levels (<20 mg/dl) and no detection of α2M by immunoelectrophoresis (defined as α2M deficiency). We also showed a relationship between serum α2M levels and acute inflammatory biomarkers in PCa patients with or without α2M deficiency. In this study, we analyzed in detail the clinicopathological characteristics and pathogenesis of α2M deficiency in androgen-dependent advanced PCa patients. In this study, 15 PCa patients were diagnosed at the Kitasato University Hospital. α2M levels were determined by laser-nephelometry and immunoelectrophoresis, and PSA levels were determined by enzyme immunoassay. IL-6 levels were measured by a specific luminescence sandwich-type enzyme-linked immunosorbent assay, and CRP levels were determined by latex nephelometry. Immunohistochemical staining for PSA in PCa specimens was also performed. The binding assay for purified α2M and PSA was analyzed by western blotting. α2M deficiency was specific for advanced PCa patients with multiple distant bone metastases. PSA was markedly detected in sera and prostate specimens of advanced PCa patients with α2M deficiency, and there was a negative correlation between serum α2M and PSA levels during the course of clinical treatment. Acute inflammatory biomarkers such as IL-6 and CRP were within reference range in α2M-deficient patients. The binding assay showed that PSA easily bound to α2M, which was detected as an approximately 800-kDa complex by western blotting. Further, genetic analysis of a α2M-deficient patient showed no mutations in the α2M gene. These results suggested that α2M deficiency develops from catabolism of α2M in androgen-dependent advanced PCa patients, and serum α2M level may be an indicator of PCa disease progression in addition to PSA level.

Levels of acute inflammatory biomarkers in advanced prostate cancer patients with α2-macroglobulin deficiency.

C-reactive protein (CRP), serum amyloid A (SAA), interleukin-6 (IL-6), α1-antitrypsin (α1AT), α1-acid glycoprotein (α1AG) and ceruloplasmin (CP) are acute inflammatory biomarkers that increase in various conditions including infection, inflammation, malignancy and tissue disturbance. In contrast, α2-macroglobulin (α2M) is involved in inflammation through its function as a carrier protein of IL-6. We had previously reported on advanced prostate cancer (PCa) patients with multiple distant bone metastases in whom serum α2M levels were markedly decreased (α2M deficiency). However, the relationship between serum levels of α2M and acute inflammatory biomarkers in PCa patients with or without α2M deficiency has not been demonstrated. In the present study, we examined serum levels of CRP, SAA, IL-6, α1AT, α1AG and CP in PCa patients with or without α2M deficiency to establish clinical significance and changes in these biomarkers during PCa disease progression. We found that upon addition of recombinant IL-6 (rIL-6) to serum from PCa patients with α2M deficiency, since a function of α2M is to bind and stabilize IL-6, the α2M-IL-6 complex and free endogenous IL-6 were not detectable. Serum levels of the α2M-independent markers, α1AT, α1AG and CP, in all PCa patients regardless of α2M deficiency were significantly higher than in healthy controls, but those of the α2M-dependent molecules, CRP, SAA and IL-6, were not increased in PCa patients with α2M deficiency. Therefore, quantitation of both α2M-dependent (CRP, SAA and IL-6) and α2M-independent (α1AT, α1AG and CP) acute inflammatory biomarkers in advanced PCa patients may be an auxiliary indicator, together with prostate-specific antigen (PSA), to monitor PCa disease progression.

Associations of IgG N-linked oligosaccharide chains and proteases in sera of prostate cancer patients with and without alpha2-macroglobulin deficiency.

We previously reported on a number of cases of metastatic prostate cancer (PCa) in which serum alpha2-macroglobulin (alpha2M) levels were markedly decreased to less than 20 mg/dl (alpha2M deficiency). All PCa patients with alpha2M deficiency had multiple bone metastases. Proteases in ten PCa patients with and without alpha2M deficiency were studied and compared against ten healthy controls in order to elucidate the relationships between changes in sugar chain structure and neoplasia. We assessed the relationship between ratios of Fr4 to Fr1 and Fr2 (Fr4/Fr1+Fr2 ratios) of oligosaccharide chains, and ratios of free prostate-specific antigen (PSA) to total PSA (F/T ratios), and serum levels of matrix-metalloproteinase-2 (MMP-2) in PCa progression. Measurement of serum alpha2M concentration was performed by laser nephelometry. Serum PSA and MMP-2 levels were determined by enzyme immunoassay and free PSA by radioimmunoassay. N-linked oligosaccharides of human serum immunoglobulin G were analyzed using fluorophore-associated carbohydrate electrophoresis. In those PCa patients with alpha2M deficiency: (a) serum alpha2M and F/T ratios were lower (P<0.05) and (b) Fr4/Fr1+Fr2 ratios and serum MMP-2 levels were higher when compared with those PCa patients without alpha2M deficiency. There was a significant correlation between Fr4/Fr1+Fr2 ratios and F/T ratios or serum MMP-2 levels in PCa with alpha2M deficiency (P<0.05). Therefore, these markers may serve as an auxiliary serum tumor marker for monitoring of the bone metastases or progression of disease in PCa.

Prognostic potential of a PSA complex in sera of prostate cancer patients with alpha2-macroglobulin deficiency.

We previously reported on a number of cases of metastatic prostate cancer (PCa) in which serum alpha2-macroglobulin (alpha2M) levels were markedly decreased to less than 20 mg/dl (alpha2M deficiency). In order to elucidate the relative proportions of free and a prostate-specific antigen (PSA) complex in PCa patients with alpha2M deficiency, we have assessed serum alpha2M and total PSA levels, and ratios of free PSA to total PSA (F/T ratios) at each stage of PCa. Moreover, the PSA reactivity profile was determined on fractionated serum specimens of PCa patients using high-performance liquid chromatography (HPLC) using a TSKG-3000 SWXL column. Measurement of alpha2M concentration was performed by laser-nephelometry. PSA levels were determined by enzyme immunoassay, free PSA by radioimmunoassay. In those PCa patients with alpha2M deficiency, serum alpha2M and F/T ratios were lower, whereas PSA levels were higher when compared with those PCa patients without alpha2M deficiency (P<0.05). PSA elution profiles on HPLC columns revealed two major peaks. The proportion of PSA-antichymotrypsin (PSA-ACT) increased, whereas the proportion of free PSA decreased in PCa patients with alpha2M deficiency as compared with those PCa patients without alpha2M deficiency. F/T ratios were significantly lower in PCa patients with alpha2M deficiency than in those PCa patients without alpha2M deficiency. PSA-ACT and F/T ratio may be useful for monitoring bone metastasis in PCa.

Differential response of a(2)-macroglobulin-deficient mice in models of lethal TNF-induced inflammation.

Tumor necrosis factor (TNF) is an essential mediator in the pathogenesis of Gram-negative septic shock. Injection of TNF into normal mice leads to systemic, lethal inflammation, which is indistinguishable from lipopolysaccharide (LPS)-induced lethal inflammation. alpha(2)-macroglobulin (A2M) is a major positive acute phase protein with broad-spectrum protease-inhibitory activity. Mouse A2M-deficient (MAM-/-) mice were significantly protected against lethal systemic inflammation induced by TNF. The protection is not due to faster clearance of the injected TNF. The induction of tolerance to TNF-induced lethality by repetitive administration of small doses of human TNF for five consecutive days was equally efficient in both mutant mice compared to wild-type mice. In D-(+)-galactosamine (GalN)-sensitized mice, TNF induces lethal inflammatory hepatitis. MAM(-/-) mice are equally sensitive to the lethal combination of TNF/GalN. Furthermore, interleukin-1-induced desensitization to TNF/GalN was not impaired in MAM(-/-) mice. We conclude that MAM plays a mediating role in TNF-induced lethal shock and that MAM deficiency does not reduce changes in efficiency of tolerance and desensitization to TNF and TNF/GalN-induced lethality, respectively.

[Immunological background of plasma protein abnormalities--the relation between inflammation and malignant neoplasm].

Mechanisms causing negative findings of serum C-reactive protein (CRP) in inflammatory disorders and malignant neoplasms were investigated. Patients with advanced prostate cancer manifesting negative CRP and alpha 2M deficiency were found. This finding suggests that alpha 2M, a carrier protein of interleukin-6, mediates CRP synthesis by the liver. Mechanism of synthesis of acute phase proteins including CRP, SAA and others was shown to be different in response to inflammation, alpha 2M-dependence in alpha 2M deficiency, the production of CRP and SAA by human hepatoma cells (HepG2) and the processes on the transcriptional activation of acute phase protein genes. In cases of prostate cancer associated with serum alpha 2M deficiency metastasis to the bones was noted. This finding suggests that alpha 2M inhibits metastasis of prostate cancer. It was suggested that the alpha 2M deficiency develops from complex formation of alpha 2M with proteases including PSA and u-PA, and accelerated catabolism of the complex rather than suppressed production of alpha 2M.

alpha2-macroglobulin- and murinoglobulin-1- deficient mice. A mouse model for acute pancreatitis.

Mice deficient in either or both mouse alpha2-macroglobulin (MAM) and murinoglobulin-1 (MUG1) were generated and proved phenotypically normal under standard conditions. Acute pancreatitis was induced with a diet deficient in choline and methionine, supplemented with ethionine. The mortality was less than 25% in wild-type mice, as opposed to at least 56% in knockout mice, and was highest (70%) in MAM-/- mice, with earliest onset at 2 days. Plasma amylase and lipase levels were increased, but pancreatic tissue appeared histologically variable in individual mice. The clinical symptoms were most severe in MAM-/- mice and, surprisingly, were not aggravated in the double knockout mice, suggesting that the lack of proteinase inhibition capacity was not the major problem. Therefore, we analyzed the expression of 21 different cytokines and polypeptide factors in the pancreas of all experimental groups of mice. Interleukin-1-receptor antagonist mRNA was consistently induced by the diet in the pancreas of MAM-/- mice, and transforming growth factor-beta, tumor necrosis factor-alpha, tumor necrosis factor-beta, beta-lymphotoxin, and interferon-gamma mRNA levels were also increased. The data demonstrate the important role of alpha2-macroglobulin (A2M) in acute pancreatitis as both a proteinase inhibitor and a cytokine carrier. Mice deficient in MAM and/or MUG thus offer new experimental models for defining in vivo the role of the macroglobulins in pancreatitis and in other normal and pathological processes.

[Concentrations of free form and complex form of prostate-specific antigen in serum of alpha 2 macroglobulin deficient patients with prostatic carcinoma].

Prostate-specific antigen (PSA) is present in blood in free form as well as in complex form with various protease inhibitors such as alpha 2 macroglobulin (alpha 2M) and alpha 1 antichymotrypsin (alpha 1 ACT). We had found that alpha 2M is deficient (below approximately 40 mg/dl) in some patients with prostatic carcinoma. Therefore, we investigated the levels of free and complex form of PSA in patients with prostatic disease and obtained the following results. The HPLC study showed that total (free plus complex) PSA level was much higher in the alpha 2M deficient patients with prostatic carcinoma stage D (n = 7, range 1,530-14,746 ng/ml, median value 6,800 ng/ml) than in the non-deficient patients with stage D (n = 16, range 121.6-4,210 ng/ml, median value 851 ng/ml). In the deficient patients, the complex of PSA with alpha 1 ACT increased extraordinarily while free PSA increased to only some extent. In the more severe cases of prostatic carcinoma, the ratio (complex/total PSA) was higher while the ratio (free/total PSA) was lower. The SDS-PAGE Western blotting showed that complex PSA increased extraordinarily and free PSA increased in sera of the deficient patients which was consistent with the HPLC results. Many bands appeared on the blotting at the positions smaller than alpha 2M molecule, which indicated that many fragments of alpha 2M were present in their sera. These bands were more intense than the bands with sera of controls or benign prostatic hypertrophy. The alpha 2M deficiency may be due to the rapid disappearance of the complex with PSA or any other prostate-originated proteases. The complex may undergo accelerated degradation or catabolism of alpha 2M.

[Studies on alpha 2 macroglobulin deficiency in association with cancer metastasis].

We found 5 cases of prostatic carcinoma with metastasis with alpha 2 macroglobulin (alpha 2 M) concentration below approximately 40 mg/dl in serum. All these patients had bone metastasis, and none of them had DIC. We found no other cases with such a low concentration of alpha 2 M. Their alpha 2 M level increased to normal level after treatment with transurethral resection of prostate or hormone agents, and the level was correlated with the clinical symptom. During the clinical course, their alpha 2 M level was negatively correlated with prostate-specific antigen (PSA) and prostatic acid phosphate (PAP). All these results suggest that alpha 2 M concentration in serum reflects the severity of prostatic carcinoma with metastasis and that alpha 2 M deficiency is an indicator of metastasis. The acute phase proteins of CRP and serum amyloid A did not increase in spite of the presence of metastasis in these patients with extremely low alpha 2 M level (< 20 mg/dl), suggesting that alpha 2 M is involved in the metabolism of these acute phase proteins. On immunohistochemical studies, their specimens of prostatic carcinoma gave positive stain for PSA and urokinase-type plasminogen activator (u-PA). Both PSA and u-PA formed a complex with alpha 2 M in vitro. The alpha 2 M deficiency in these patients might be due to the complex formation between alpha 2 M and these prostate-originated proteases and to the rapid disappearance of the complex.

Targeted inactivation of the mouse alpha 2-macroglobulin gene.

The mouse alpha 2-macroglobulin gene was inactivated in embryonic stem cells by homologous recombination. Liver alpha 2-macroglobulin mRNA and plasma protein was absent in homozygotes and reduced to 50% in heterozygotes. alpha 2-Macroglobulin-deficient mice were viable and produced normally sized litters with normal sex ratio over 3 generations. Characterization of adult homozygotes included diets with different fat content, treatments with endotoxin, bleomycin, carbon tetrachloride, and ethionine to test for immune system, lung, liver, and pancreas toxicity, respectively. Knock-out mice were more resistant to endotoxin but more sensitive to a choline-free diet supplemented with ethionine. Regulation of murinoglobulin mRNA expression during pregnancy was analyzed as a possible back-up mechanism for the deficiency in alpha 2-macroglobulin. In addition, expression of mRNA was studied, coding for alpha 2-macroglobulin receptor/lipoprotein receptor-related protein, low density lipoprotein receptor, and very low density lipoprotein receptor and for some common ligands, i.e. apolipoprotein E, lipoprotein lipase, and the 44-kDa heparin binding protein. Their differential regulation in the knock-out mice relative to C57B1 mice was evident and is discussed. The impressive 15-fold increase in maternal liver murinoglobulin mRNA at partum in the knock-out mice indicated increased consumption, compared to only 4-fold in normal mice. Thus, murinoglobulin appears as the major proteinase inhibitor around partum, obviously solicited to a much greater extend in alpha 2-macroglobulin-deficient mice.

Open-heart surgery in a patient with heterozygous alpha 2-antiplasmin deficiency. Perioperative strategies in the first reported case.

alpha 2-Antiplasmin deficiency is a serious coagulation disorder that results in unrestrained fibrinolytic activity. Clinically, it is manifested by instability of the fibrin hemostatic plug and prolonged or delayed bleeding, which is more serious in patients who are homozygous for this trait. A patient scheduled for aortic valve replacement and coronary bypass presented with a history of repeated episodes of postoperative bleeding. Hemostatic laboratory evaluation revealed that the patient had the heterozygous form of alpha 2-antiplasmin deficiency with a serum concentration of 52 percent (normal, greater than 65 percent of the activity of pooled plasma). He underwent preoperative plasmapheresis with administration of 3,000 ml of fresh frozen plasma, which resulted in an increase in the preoperative level of alpha 2-antiplasmin to 78 percent. Although postoperative blood loss was greater than normal, it was easily managed. Preoperative identification of this rare coagulation abnormality permitted appropriate treatment and probably prevented a postoperative death from hemorrhage.

Impaired secretion of mutant alpha 2-plasmin inhibitor (alpha 2 PI-Nara) from COS-7 and HepG2 cells: molecular and cellular basis for hereditary deficiency of alpha 2-plasmin inhibitor.

The elongated mutant of alpha 2-plasmin inhibitor (alpha 2 PI) designated as alpha 2 PI-Nara is caused by a frameshift mutation found near the 3' end of the coding region of the alpha 2 PI gene. To elucidate the mechanism by which this molecular abnormality leads to alpha 2 PI deficiency in plasma, we transfected an expression plasmid for alpha 2 PI-Nara into a monkey kidney cell line COS-7 or human hepatoma cell line HepG2 synthesizing alpha 2 PI, and analyzed the secretory process of the expressed alpha 2 PI-Nara by radioimmunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. The results obtained showed that the recombinant alpha 2 PI-Nara was retained within the cells for prolonged periods as an endoglycosidase H-sensitive precursor form, and only a small portion of the recombinant protein was secreted into the medium as a neuraminidase-sensitive mature form. These results suggest that instead of being secreted from the cells, most of the alpha 2 PI-Nara undergoes degradation within the cells while its transport is retarded in the intracellular secretory pathway; thus, alpha 2 PI-Nara should lead to the alpha 2 PI deficiency primarily by causing a block in the intracellular transport from the endoplasmic reticulum to the Golgi complex.

[Gas exchange, microcirculation and blood kinins in patients with chronic nonspecific lung diseases]. / Gazoobmen, mikrotsirkuliatsiia i kininy krovi u bol'nykh khronicheskimi nespetsificheskimi zabolevaniiami legkikh.

The state of gas exchange, microcirculation (MC) and blood kinin was examined in 212 patients with chronic non-specific pulmonary diseases (197 with respiratory insufficiency, 15 without it). Noticeable activation of blood kinins with a rise of kallikrein, a decrease in alpha 2-macroglobulin and kinase activity were revealed in patients with respiratory insufficiency (RI), degree I and II, expressed in MC disorder with perivascular and intravascular changes. Kininogesis suppression with a decrease in all indices was noted in patients with RI, degree III, with progressive hypoxemia, hypercapnia, MC disorder in all the links. An insignificant positive time course in the state of gas exchange, MC and blood kinins after therapy was indicative of insufficient efficacy of multiple modality therapy and permitted recommendation of drugs correcting disorders in the above systems.

New and rapid functional assay for C1 inhibitor in human plasma.

C1 inhibitor (C1-INH) and alpha 2-macroglobulin (alpha 2M) account for over 90% of the inactivation of purified plasma kallikrein by normal human plasma. The rate of kallikrein inactivation is also dependent on the presence of high molecular weight kininogen (HMWK), which forms a reversible complex with kallikrein protecting the active site of the enzyme against inhibitors. By selectively inactivating alpha 2M with methylamine, and eliminating the protective effect of HMWK by dilution, the inactivation of kallikrein by plasma became almost exclusively dependent on C1-INH. Functional C1 inhibitor was assessed by measuring the pseudo-first-order rate constant for the inactivation of kallikrein by diluted methylamine-treated plasma in 29 individuals, including 11 controls, 11 oral contraceptive users, 5 patients with classical hereditary angioedema (HAE), and 2 patients with variant HAE. Over a wide range of concentrations, an excellent correlation (r = 0.90) was observed between functional and antigenic C1-INH among controls, oral contraceptive users, and patients with classical HAE. This new functional assay for C1-INH can be performed in less than 3 hr with commercially available reagents. Therefore, this assay will be helpful for the diagnosis and management of conditions associated with the deficiency of C1-INH, such as HAE.

Cardiovascular hemodynamics after opsonic alpha-2-surface binding glycoprotein therapy in injured patients.

Depression of reticuloendothelial (RE) phagocytic function has been clearly documented following trauma and operation. This phagocytic failure is mediated in part by depletion of an opsonic glycoprotein. Depletion of this opsonic protein may result in prolonged blood retention of potentially harmful particulates that may interfere with the microcirculation and may possibly result in altered organ function. Isolation and identification of this opsonic protein has led to the finding of the identity between opsonic glycoprotein and cold insoluble globulin (CIg) or so-called plasma fibronectin. Since CIg is concentrated in cryoprecipitate, this blood component was used as a readily available source of opsonic protein for replacement studies. Nine patients were studied following a 1-hour infusion of cryoprecipitate obtained from 10 units of plasma and suspended in a volume of 250 ml. Both the pulmonary shunt fraction and the fraction of dead space ventilation decreased significantly (P = 0.02) after cryoprecipitate administration. Limb blood flow (P = 0.001), limb oxygen consumption (P = 0.001), and reactive hyperemia of the limb (P = 0.05) increased significantly following cryoprecipitate infusion. Cardiac output, total oxygen consumption did not change consistently. The data demonstrate that the infusion of cryoprecipitate resulted in improved pulmonary and microcirculatory function--possibly due to opsonic glycoprotein replacement.